Methods and compositions for diagnosis and prognosis of renal injury and renal failure

ABSTRACT

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects suffering from or suspected of having a renal injury. In particular, the invention relates to using assays that detect Chitinase-3-like protein 1 as diagnostic and prognostic biomarker assays in renal injuries.

This application claims the benefit of U.S. Provisional Application No.62/093,939 filed Dec. 18, 2014, which is hereby incorporated in itsentirety including all tables, figures, and claims.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

The kidney is responsible for water and solute excretion from the body.Its functions include maintenance of acid-base balance, regulation ofelectrolyte concentrations, control of blood volume, and regulation ofblood pressure. As such, loss of kidney function through injury and/ordisease results in substantial morbidity and mortality. A detaileddiscussion of renal injuries is provided in Harrison's Principles ofInternal Medicine, 17^(th) Ed., McGraw Hill, New York, pages 1741-1830,which are hereby incorporated by reference in their entirety. Renaldisease and/or injury may be acute or chronic. Acute and chronic kidneydisease are described as follows (from Current Medical Diagnosis &Treatment 2008, 47^(th) Ed, McGraw Hill, New York, pages 785-815, whichare hereby incorporated by reference in their entirety): “Acute renalfailure is worsening of renal function over hours to days, resulting inthe retention of nitrogenous wastes (such as urea nitrogen) andcreatinine in the blood. Retention of these substances is calledazotemia. Chronic renal failure (chronic kidney disease) results from anabnormal loss of renal function over months to years”.

Acute renal failure (ARF, also known as acute kidney injury, or AKI) isan abrupt (typically detected within about 48 hours to 1 week) reductionin glomerular filtration. This loss of filtration capacity results inretention of nitrogenous (urea and creatinine) and non-nitrogenous wasteproducts that are normally excreted by the kidney, a reduction in urineoutput, or both. It is reported that ARF complicates about 5% ofhospital admissions, 4-15% of cardiopulmonary bypass surgeries, and upto 30% of intensive care admissions. ARF may be categorized as prerenal,intrinsic renal, or postrenal in causation. Intrinsic renal disease canbe further divided into glomerular, tubular, interstitial, and vascularabnormalities. Major causes of ARF are described in the following table,which is adapted from the Merck Manual, 17^(th) ed., Chapter 222, andwhich is hereby incorporated by reference in their entirety:

Type Risk Factors Prerenal ECF volume depletion Excessive diuresis,hemorrhage, GI losses, loss of intravascular fluid into theextravascular space (due to ascites, peritonitis, pancreatitis, orburns), loss of skin and mucus membranes, renal salt- and water-wastingstates Low cardiac output Cardiomyopathy, MI, cardiac tamponade,pulmonary embolism, pulmonary hypertension, positive-pressure mechanicalventilation Low systemic vascular Septic shock, liver failure,antihypertensive drugs resistance Increased renal vascular NSAIDs,cyclosporines, tacrolimus, hypercalcemia, resistance anaphylaxis,anesthetics, renal artery obstruction, renal vein thrombosis, sepsis,hepatorenal syndrome Decreased efferent ACE inhibitors or angiotensin IIreceptor blockers arteriolar tone (leading to decreased GFR from reducedglomerular transcapillary pressure, especially in patients withbilateral renal artery stenosis) Intrinsic Renal Acute tubular injuryIschemia (prolonged or severe prerenal state): surgery, hemorrhage,arterial or venous obstruction; Toxins: NSAIDs, cyclosporines,tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin,myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrastagents, streptozotocin Acute glomerulonephritis ANCA-associated:Crescentic glomerulonephritis, polyarteritis nodosa, Wegener'sgranulomatosis; Anti- GBM glomerulonephritis: Goodpasture's syndrome;Immune-complex: Lupus glomerulonephritis, postinfectiousglomerulonephritis, cryoglobulinemic glomerulonephritis Acutetubulointerstitial Drug reaction (eg, β-lactams, NSAIDs, sulfonamides,nephritis ciprofloxacin, thiazide diuretics, furosemide, phenytoin,allopurinol, pyelonephritis, papillary necrosis Acute vascularVasculitis, malignant hypertension, thrombotic nephropathymicroangiopathies, scleroderma, atheroembolism Infiltrative diseasesLymphoma, sarcoidosis, leukemia Postrenal Tubular precipitation Uricacid (tumor lysis), sulfonamides, triamterene, acyclovir, indinavir,methotrexate, ethylene glycol ingestion, myeloma protein, myoglobinUreteral obstruction Intrinsic: Calculi, clots, sloughed renal tissue,fungus ball, edema, malignancy, congenital defects; Extrinsic:Malignancy, retroperitoneal fibrosis, ureteral trauma during surgery orhigh impact injury Bladder obstruction Mechanical: Benign prostatichyperplasia, prostate cancer, bladder cancer, urethral strictures,phimosis, paraphimosis, urethral valves, obstructed indwelling urinarycatheter; Neurogenic: Anticholinergic drugs, upper or lower motor neuronlesion

In the case of ischemic ARF, the course of the disease may be dividedinto four phases. During an initiation phase, which lasts hours to days,reduced perfusion of the kidney is evolving into injury. Glomerularultrafiltration reduces, the flow of filtrate is reduced due to debriswithin the tubules, and back leakage of filtrate through injuredepithelium occurs. Renal injury can be mediated during this phase byreperfusion of the kidney. Initiation is followed by an extension phasewhich is characterized by continued ischemic injury and inflammation andmay involve endothelial damage and vascular congestion. During themaintenance phase, lasting from 1 to 2 weeks, renal cell injury occurs,and glomerular filtration and urine output reaches a minimum. A recoveryphase can follow in which the renal epithelium is repaired and GFRgradually recovers. Despite this, the survival rate of subjects with ARFmay be as low as about 60%.

Acute kidney injury caused by radiocontrast agents (also called contrastmedia) and other nephrotoxins such as cyclosporine, antibioticsincluding aminoglycosides and anticancer drugs such as cisplatinmanifests over a period of days to about a week. Contrast inducednephropathy (CIN, which is AKI caused by radiocontrast agents) isthought to be caused by intrarenal vasoconstriction (leading to ischemicinjury) and from the generation of reactive oxygen species that aredirectly toxic to renal tubular epithelial cells. CIN classicallypresents as an acute (onset within 24-48 h) but reversible (peak 3-5days, resolution within 1 week) rise in blood urea nitrogen and serumcreatinine.

A commonly reported criteria for defining and detecting AKI is an abrupt(typically within about 2-7 days or within a period of hospitalization)elevation of serum creatinine. Although the use of serum creatinineelevation to define and detect AKI is well established, the magnitude ofthe serum creatinine elevation and the time over which it is measured todefine AKI varies considerably among publications. Traditionally,relatively large increases in serum creatinine such as 100%, 200%, anincrease of at least 100% to a value over 2 mg/dL and other definitionswere used to define AKI. However, the recent trend has been towardsusing smaller serum creatinine rises to define AKI. The relationshipbetween serum creatinine rise, AKI and the associated health risks arereviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14:265-270,2005 and Chertow et al, J Am Soc Nephrol 16: 3365-3370, 2005, which,with the references listed therein, are hereby incorporated by referencein their entirety. As described in these publications, acute worseningrenal function (AKI) and increased risk of death and other detrimentaloutcomes are now known to be associated with very small increases inserum creatinine. These increases may be determined as a relative(percent) value or a nominal value. Relative increases in serumcreatinine as small as 20% from the pre-injury value have been reportedto indicate acutely worsening renal function (AKI) and increased healthrisk, but the more commonly reported value to define AKI and increasedhealth risk is a relative increase of at least 25%. Nominal increases assmall as 0.3 mg/dL, 0.2 mg/dL or even 0.1 mg/dL have been reported toindicate worsening renal function and increased risk of death. Varioustime periods for the serum creatinine to rise to these threshold valueshave been used to define AKI, for example, ranging from 2 days, 3 days,7 days, or a variable period defined as the time the patient is in thehospital or intensive care unit. These studies indicate there is not aparticular threshold serum creatinine rise (or time period for the rise)for worsening renal function or AKI, but rather a continuous increase inrisk with increasing magnitude of serum creatinine rise.

One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605, 2004, herebyincorporated by reference in its entirety) investigated both increasesand decreases in serum creatinine. Patients with a mild fall in serumcreatinine of −0.1 to −0.3 mg/dL following heart surgery had the lowestmortality rate. Patients with a larger fall in serum creatinine (morethan or equal to −0.4 mg/dL) or any increase in serum creatinine had alarger mortality rate. These findings caused the authors to concludethat even very subtle changes in renal function (as detected by smallcreatinine changes within 48 hours of surgery) seriously effectpatient's outcomes. In an effort to reach consensus on a unifiedclassification system for using serum creatinine to define AKI inclinical trials and in clinical practice, Bellomo et al., Crit Care.8(4):R204-12, 2004, which is hereby incorporated by reference in itsentirety, proposes the following classifications for stratifying AKIpatients:

“Risk”: serum creatinine increased 1.5 fold from baseline OR urineproduction of <0.5 ml/kg body weight/hr for 6 hours;“Injury”: serum creatinine increased 2.0 fold from baseline OR urineproduction <0.5 ml/kg/hr for 12 h;“Failure”: serum creatinine increased 3.0 fold from baseline ORcreatinine >355 μmol/l (with a rise of >44) or urine output below 0.3ml/kg/hr for 24 h or anuria for at least 12 hours;And included two clinical outcomes:“Loss”: persistent need for renal replacement therapy for more than fourweeks.“ESRD”: end stage renal disease—the need for dialysis for more than 3months.These criteria are called the RIFLE criteria, which provide a usefulclinical tool to classify renal status. As discussed in Kellum, Crit.Care Med. 36: S141-45, 2008 and Ricci et al., Kidney Int. 73, 538-546,2008, each hereby incorporated by reference in its entirety, the RIFLEcriteria provide a uniform definition of AKI which has been validated innumerous studies.

More recently, Mehta et al., Crit. Care 11:R31 (doi:10.1186.cc5713),2007, hereby incorporated by reference in its entirety, proposes thefollowing similar classifications for stratifying AKI patients, whichhave been modified from RIFLE:

“Stage I”: increase in serum creatinine of more than or equal to 0.3mg/dL (≧26.4 μmol/L) or increase to more than or equal to 150%(1.5-fold) from baseline OR urine output less than 0.5 mL/kg per hourfor more than 6 hours;“Stage II”: increase in serum creatinine to more than 200% (>2-fold)from baseline OR urine output less than 0.5 mL/kg per hour for more than12 hours;“Stage III”: increase in serum creatinine to more than 300% (>3-fold)from baseline OR serum creatinine ≧354 μmol/L accompanied by an acuteincrease of at least 44 μmol/L OR urine output less than 0.3 mL/kg perhour for 24 hours or anuria for 12 hours.

The CIN Consensus Working Panel (McCollough et al, Rev Cardiovasc Med.2006; 7(4):177-197, hereby incorporated by reference in its entirety)uses a serum creatinine rise of 25% to define Contrast inducednephropathy (which is a type of AKI). Although various groups proposeslightly different criteria for using serum creatinine to detect AKI,the consensus is that small changes in serum creatinine, such as 0.3mg/dL or 25%, are sufficient to detect AKI (worsening renal function)and that the magnitude of the serum creatinine change is an indicator ofthe severity of the AKI and mortality risk.

Although serial measurement of serum creatinine over a period of days isan accepted method of detecting and diagnosing AKI and is considered oneof the most important tools to evaluate AKI patients, serum creatinineis generally regarded to have several limitations in the diagnosis,assessment and monitoring of AKI patients. The time period for serumcreatinine to rise to values (e.g., a 0.3 mg/dL or 25% rise) considereddiagnostic for AKI can be 48 hours or longer depending on the definitionused. Since cellular injury in AKI can occur over a period of hours,serum creatinine elevations detected at 48 hours or longer can be a lateindicator of injury, and relying on serum creatinine can thus delaydiagnosis of AKI. Furthermore, serum creatinine is not a good indicatorof the exact kidney status and treatment needs during the most acutephases of AKI when kidney function is changing rapidly. Some patientswith AKI will recover fully, some will need dialysis (either short termor long term) and some will have other detrimental outcomes includingdeath, major adverse cardiac events and chronic kidney disease. Becauseserum creatinine is a marker of filtration rate, it does notdifferentiate between the causes of AKI (pre-renal, intrinsic renal,post-renal obstruction, atheroembolic, etc) or the category or locationof injury in intrinsic renal disease (for example, tubular, glomerularor interstitial in origin). Urine output is similarly limited, Knowingthese things can be of vital importance in managing and treatingpatients with AKI.

These limitations underscore the need for better methods to detect andassess AKI, particularly in the early and subclinical stages, but alsoin later stages when recovery and repair of the kidney can occur.Furthermore, there is a need to better identify patients who are at riskof having an AKI.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions forevaluating renal function in a subject. As described herein, measurementof Chitinase-3-like protein 1 (referred to herein as a “kidney injurymarker”) can be used for diagnosis, prognosis, risk stratification,staging, monitoring, categorizing and determination of further diagnosisand treatment regimens in subjects suffering or at risk of sufferingfrom an injury to renal function, reduced renal function, and/or acuterenal failure (also called acute kidney injury).

Chitinase-3-like protein 1 may be used, individually or in panelscomprising a plurality of kidney injury markers, for risk stratification(that is, to identify subjects at risk for a future injury to renalfunction, for future progression to reduced renal function, for futureprogression to ARF, for future improvement in renal function, etc.); fordiagnosis of existing disease (that is, to identify subjects who havesuffered an injury to renal function, who have progressed to reducedrenal function, who have progressed to ARF, etc.); for monitoring fordeterioration or improvement of renal function; and for predicting afuture medical outcome, such as improved or worsening renal function, adecreased or increased mortality risk, a decreased or increased riskthat a subject will require renal replacement therapy (i.e.,hemodialysis, peritoneal dialysis, hemofiltration, and/or renaltransplantation, a decreased or increased risk that a subject willrecover from an injury to renal function, a decreased or increased riskthat a subject will recover from ARF, a decreased or increased risk thata subject will progress to end stage renal disease, a decreased orincreased risk that a subject will progress to chronic renal failure, adecreased or increased risk that a subject will suffer rejection of atransplanted kidney, etc.

In a first aspect, the present invention relates to methods forevaluating renal status in a subject. These methods comprise performingan assay method that is configured to detect Chitinase-3-like protein 1in a body fluid sample obtained from the subject. The assay result(s),for example a measured concentration of Chitinase-3-like protein 1,is/are then correlated to the renal status of the subject. Thiscorrelation to renal status may include correlating the assay result(s)to one or more of risk stratification, diagnosis, prognosis, staging,classifying and monitoring of the subject as described herein. Thus, thepresent invention utilizes one or more kidney injury markers of thepresent invention for the evaluation of renal injury.

In certain embodiments, the methods for evaluating renal statusdescribed herein are methods for risk stratification of the subject;that is, assigning a likelihood of one or more future changes in renalstatus to the subject. In these embodiments, the assay result(s) is/arecorrelated to one or more such future changes. The following arepreferred risk stratification embodiments.

In preferred risk stratification embodiments, these methods comprisedetermining a subject's risk for a future injury to renal function, andthe assay result(s) is/are correlated to a likelihood of such a futureinjury to renal function. For example, the measured concentration(s) mayeach be compared to a threshold value. For a “positive going” kidneyinjury marker, an increased likelihood of suffering a future injury torenal function is assigned to the subject when the measuredconcentration is above the threshold, relative to a likelihood assignedwhen the measured concentration is below the threshold. For a “negativegoing” kidney injury marker, an increased likelihood of suffering afuture injury to renal function is assigned to the subject when themeasured concentration is below the threshold, relative to a likelihoodassigned when the measured concentration is above the threshold.

In other preferred risk stratification embodiments, these methodscomprise determining a subject's risk for future reduced renal function,and the assay result(s) is/are correlated to a likelihood of suchreduced renal function. For example, the measured concentrations mayeach be compared to a threshold value. For a “positive going” kidneyinjury marker, an increased likelihood of suffering a future reducedrenal function is assigned to the subject when the measuredconcentration is above the threshold, relative to a likelihood assignedwhen the measured concentration is below the threshold. For a “negativegoing” kidney injury marker, an increased likelihood of future reducedrenal function is assigned to the subject when the measuredconcentration is below the threshold, relative to a likelihood assignedwhen the measured concentration is above the threshold.

In still other preferred risk stratification embodiments, these methodscomprise determining a subject's likelihood for a future improvement inrenal function, and the assay result(s) is/are correlated to alikelihood of such a future improvement in renal function. For example,the measured concentration(s) may each be compared to a threshold value.For a “positive going” kidney injury marker, an increased likelihood ofa future improvement in renal function is assigned to the subject whenthe measured concentration is below the threshold, relative to alikelihood assigned when the measured concentration is above thethreshold. For a “negative going” kidney injury marker, an increasedlikelihood of a future improvement in renal function is assigned to thesubject when the measured concentration is above the threshold, relativeto a likelihood assigned when the measured concentration is below thethreshold.

In yet other preferred risk stratification embodiments, these methodscomprise determining a subject's risk for progression to ARF, and theresult(s) is/are correlated to a likelihood of such progression to ARF.For example, the measured concentration(s) may each be compared to athreshold value. For a “positive going” kidney injury marker, anincreased likelihood of progression to ARF is assigned to the subjectwhen the measured concentration is above the threshold, relative to alikelihood assigned when the measured concentration is below thethreshold. For a “negative going” kidney injury marker, an increasedlikelihood of progression to ARF is assigned to the subject when themeasured concentration is below the threshold, relative to a likelihoodassigned when the measured concentration is above the threshold.

And in other preferred risk stratification embodiments, these methodscomprise determining a subject's outcome risk, and the assay result(s)is/are correlated to a likelihood of the occurrence of a clinicaloutcome related to a renal injury suffered by the subject. For example,the measured concentration(s) may each be compared to a threshold value.For a “positive going” kidney injury marker, an increased likelihood ofone or more of: acute kidney injury, progression to a worsening stage ofAKI, mortality, a requirement for renal replacement therapy, arequirement for withdrawal of renal toxins, end stage renal disease,heart failure, stroke, myocardial infarction, progression to chronickidney disease, etc., is assigned to the subject when the measuredconcentration is above the threshold, relative to a likelihood assignedwhen the measured concentration is below the threshold. For a “negativegoing” kidney injury marker, an increased likelihood of one or more of:acute kidney injury, progression to a worsening stage of AKI, mortality,a requirement for renal replacement therapy, a requirement forwithdrawal of renal toxins, end stage renal disease, heart failure,stroke, myocardial infarction, progression to chronic kidney disease,etc., is assigned to the subject when the measured concentration isbelow the threshold, relative to a likelihood assigned when the measuredconcentration is above the threshold.

In such risk stratification embodiments, preferably the likelihood orrisk assigned is that an event of interest is more or less likely tooccur within 180 days of the time at which the body fluid sample isobtained from the subject. In particularly preferred embodiments, thelikelihood or risk assigned relates to an event of interest occurringwithin a shorter time period such as 18 months, 120 days, 90 days, 60days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 days, 96 hours, 72hours, 48 hours, 36 hours, 24 hours, 12 hours, or less. A risk at 0hours of the time at which the body fluid sample is obtained from thesubject is equivalent to diagnosis of a current condition.

In preferred risk stratification embodiments, the subject is selectedfor risk stratification based on the pre-existence in the subject of oneor more known risk factors for prerenal, intrinsic renal, or postrenalARF. For example, a subject undergoing or having undergone majorvascular surgery, coronary artery bypass, or other cardiac surgery; asubject having pre-existing congestive heart failure, preeclampsia,eclampsia, diabetes mellitus, hypertension, coronary artery disease,proteinuria, renal insufficiency, glomerular filtration below the normalrange, cirrhosis, serum creatinine above the normal range, or sepsis; ora subject exposed to NSAIDs, cyclosporines, tacrolimus, aminoglycosides,foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavymetals, methotrexate, radiopaque contrast agents, or streptozotocin areall preferred subjects for monitoring risks according to the methodsdescribed herein. This list is not meant to be limiting. By“pre-existence” in this context is meant that the risk factor exists atthe time the body fluid sample is obtained from the subject. Inparticularly preferred embodiments, a subject is chosen for riskstratification based on an existing diagnosis of injury to renalfunction, reduced renal function, or ARF.

In other embodiments, the methods for evaluating renal status describedherein are methods for diagnosing a renal injury in the subject; thatis, assessing whether or not a subject has suffered from an injury torenal function, reduced renal function, or ARF. In these embodiments,the assay result(s), for example a measured concentration ofChitinase-3-like protein 1, is/are correlated to the occurrence ornonoccurrence of a change in renal status. The following are preferreddiagnostic embodiments.

In preferred diagnostic embodiments, these methods comprise diagnosingthe occurrence or nonoccurrence of an injury to renal function, and theassay result(s) is/are correlated to the occurrence or nonoccurrence ofsuch an injury. For example, each of the measured concentration(s) maybe compared to a threshold value. For a positive going marker, anincreased likelihood of the occurrence of an injury to renal function isassigned to the subject when the measured concentration is above thethreshold (relative to the likelihood assigned when the measuredconcentration is below the threshold); alternatively, when the measuredconcentration is below the threshold, an increased likelihood of thenonoccurrence of an injury to renal function may be assigned to thesubject (relative to the likelihood assigned when the measuredconcentration is above the threshold). For a negative going marker, anincreased likelihood of the occurrence of an injury to renal function isassigned to the subject when the measured concentration is below thethreshold (relative to the likelihood assigned when the measuredconcentration is above the threshold); alternatively, when the measuredconcentration is above the threshold, an increased likelihood of thenonoccurrence of an injury to renal function may be assigned to thesubject (relative to the likelihood assigned when the measuredconcentration is below the threshold).

In other preferred diagnostic embodiments, these methods comprisediagnosing the occurrence or nonoccurrence of reduced renal function,and the assay result(s) is/are correlated to the occurrence ornonoccurrence of an injury causing reduced renal function. For example,each of the measured concentration(s) may be compared to a thresholdvalue. For a positive going marker, an increased likelihood of theoccurrence of an injury causing reduced renal function is assigned tothe subject when the measured concentration is above the threshold(relative to the likelihood assigned when the measured concentration isbelow the threshold); alternatively, when the measured concentration isbelow the threshold, an increased likelihood of the nonoccurrence of aninjury causing reduced renal function may be assigned to the subject(relative to the likelihood assigned when the measured concentration isabove the threshold). For a negative going marker, an increasedlikelihood of the occurrence of an injury causing reduced renal functionis assigned to the subject when the measured concentration is below thethreshold (relative to the likelihood assigned when the measuredconcentration is above the threshold); alternatively, when the measuredconcentration is above the threshold, an increased likelihood of thenonoccurrence of an injury causing reduced renal function may beassigned to the subject (relative to the likelihood assigned when themeasured concentration is below the threshold).

In yet other preferred diagnostic embodiments, these methods comprisediagnosing the occurrence or nonoccurrence of ARF, and the assayresult(s) is/are correlated to the occurrence or nonoccurrence of aninjury causing ARF. For example, each of the measured concentration(s)may be compared to a threshold value. For a positive going marker, anincreased likelihood of the occurrence of ARF is assigned to the subjectwhen the measured concentration is above the threshold (relative to thelikelihood assigned when the measured concentration is below thethreshold); alternatively, when the measured concentration is below thethreshold, an increased likelihood of the nonoccurrence of ARF may beassigned to the subject (relative to the likelihood assigned when themeasured concentration is above the threshold). For a negative goingmarker, an increased likelihood of the occurrence of ARF is assigned tothe subject when the measured concentration is below the threshold(relative to the likelihood assigned when the measured concentration isabove the threshold); alternatively, when the measured concentration isabove the threshold, an increased likelihood of the nonoccurrence of ARFmay be assigned to the subject (relative to the likelihood assigned whenthe measured concentration is below the threshold).

In still other preferred diagnostic embodiments, these methods comprisediagnosing a subject as being in need of renal replacement therapy, andthe assay result(s) is/are correlated to a need for renal replacementtherapy. For example, each of the measured concentration(s) may becompared to a threshold value. For a positive going marker, an increasedlikelihood of the occurrence of an injury creating a need for renalreplacement therapy is assigned to the subject when the measuredconcentration is above the threshold (relative to the likelihoodassigned when the measured concentration is below the threshold);alternatively, when the measured concentration is below the threshold,an increased likelihood of the nonoccurrence of an injury creating aneed for renal replacement therapy may be assigned to the subject(relative to the likelihood assigned when the measured concentration isabove the threshold). For a negative going marker, an increasedlikelihood of the occurrence of an injury creating a need for renalreplacement therapy is assigned to the subject when the measuredconcentration is below the threshold (relative to the likelihoodassigned when the measured concentration is above the threshold);alternatively, when the measured concentration is above the threshold,an increased likelihood of the nonoccurrence of an injury creating aneed for renal replacement therapy may be assigned to the subject(relative to the likelihood assigned when the measured concentration isbelow the threshold).

In still other preferred diagnostic embodiments, these methods comprisediagnosing a subject as being in need of renal transplantation, and theassay result(s0 is/are correlated to a need for renal transplantation.For example, each of the measured concentration(s) may be compared to athreshold value. For a positive going marker, an increased likelihood ofthe occurrence of an injury creating a need for renal transplantation isassigned to the subject when the measured concentration is above thethreshold (relative to the likelihood assigned when the measuredconcentration is below the threshold); alternatively, when the measuredconcentration is below the threshold, an increased likelihood of thenonoccurrence of an injury creating a need for renal transplantation maybe assigned to the subject (relative to the likelihood assigned when themeasured concentration is above the threshold). For a negative goingmarker, an increased likelihood of the occurrence of an injury creatinga need for renal transplantation is assigned to the subject when themeasured concentration is below the threshold (relative to thelikelihood assigned when the measured concentration is above thethreshold); alternatively, when the measured concentration is above thethreshold, an increased likelihood of the nonoccurrence of an injurycreating a need for renal transplantation may be assigned to the subject(relative to the likelihood assigned when the measured concentration isbelow the threshold).

In still other embodiments, the methods for evaluating renal statusdescribed herein are methods for monitoring a renal injury in thesubject; that is, assessing whether or not renal function is improvingor worsening in a subject who has suffered from an injury to renalfunction, reduced renal function, or ARF. In these embodiments, theassay result(s), for example a measured concentration ofChitinase-3-like protein 1, is/are correlated to the occurrence ornonoccurrence of a change in renal status. The following are preferredmonitoring embodiments.

In preferred monitoring embodiments, these methods comprise monitoringrenal status in a subject suffering from an injury to renal function,and the assay result(s) is/are correlated to the occurrence ornonoccurrence of a change in renal status in the subject. For example,the measured concentration(s) may be compared to a threshold value. Fora positive going marker, when the measured concentration is above thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In other preferred monitoring embodiments, these methods comprisemonitoring renal status in a subject suffering from reduced renalfunction, and the assay result(s) is/are correlated to the occurrence ornonoccurrence of a change in renal status in the subject. For example,the measured concentration(s) may be compared to a threshold value. Fora positive going marker, when the measured concentration is above thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In yet other preferred monitoring embodiments, these methods comprisemonitoring renal status in a subject suffering from acute renal failure,and the assay result(s) is/are correlated to the occurrence ornonoccurrence of a change in renal status in the subject. For example,the measured concentration(s) may be compared to a threshold value. Fora positive going marker, when the measured concentration is above thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In other additional preferred monitoring embodiments, these methodscomprise monitoring renal status in a subject at risk of an injury torenal function due to the pre-existence of one or more known riskfactors for prerenal, intrinsic renal, or postrenal ARF, and the assayresult(s) is/are correlated to the occurrence or nonoccurrence of achange in renal status in the subject. For example, the measuredconcentration(s) may be compared to a threshold value. For a positivegoing marker, when the measured concentration is above the threshold, aworsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In still other embodiments, the methods for evaluating renal statusdescribed herein are methods for classifying a renal injury in thesubject; that is, determining whether a renal injury in a subject isprerenal, intrinsic renal, or postrenal; and/or further subdividingthese classes into subclasses such as acute tubular injury, acuteglomerulonephritis acute tubulointerstitial nephritis, acute vascularnephropathy, or infiltrative disease; and/or assigning a likelihood thata subject will progress to a particular RIFLE stage. In theseembodiments, the assay result(s), for example a measured concentrationof Chitinase-3-like protein 1, is/are correlated to a particular classand/or subclass. The following are preferred classification embodiments.

In preferred classification embodiments, these methods comprisedetermining whether a renal injury in a subject is prerenal, intrinsicrenal, or postrenal; and/or further subdividing these classes intosubclasses such as acute tubular injury, acute glomerulonephritis acutetubulointerstitial nephritis, acute vascular nephropathy, orinfiltrative disease; and/or assigning a likelihood that a subject willprogress to a particular RIFLE stage, and the assay result(s) is/arecorrelated to the injury classification for the subject. For example,the measured concentration may be compared to a threshold value, andwhen the measured concentration is above the threshold, a particularclassification is assigned; alternatively, when the measuredconcentration is below the threshold, a different classification may beassigned to the subject.

A variety of methods may be used by the skilled artisan to arrive at adesired threshold value for use in these methods. For example, thethreshold value may be determined from a population of normal subjectsby selecting a concentration representing the 75^(th), 85^(th), 90^(th),95^(th), or 99^(th) percentile of a kidney injury marker measured insuch normal subjects. Alternatively, the threshold value may bedetermined from a “diseased” population of subjects, e.g., thosesuffering from an injury or having a predisposition for an injury (e.g.,progression to ARF or some other clinical outcome such as death,dialysis, renal transplantation, etc.), by selecting a concentrationrepresenting the 75^(th), 85^(th), 90^(th), 95^(th), or 99^(th)percentile of a kidney injury marker measured in such subjects. Inanother alternative, the threshold value may be determined from a priormeasurement of a kidney injury marker in the same subject; that is, atemporal change in the level of a kidney injury marker in the subjectmay be used to assign risk to the subject.

The foregoing discussion is not meant to imply, however, that the kidneyinjury markers of the present invention must be compared tocorresponding individual thresholds. Methods for combining assay resultscan comprise the use of multivariate logistical regression, loglinearmodeling, neural network analysis, n-of-m analysis, decision treeanalysis, calculating ratios of markers, etc. This list is not meant tobe limiting. In these methods, a composite result which is determined bycombining individual markers may be treated as if it is itself a marker;that is, a threshold may be determined for the composite result asdescribed herein for individual markers, and the composite result for anindividual patient compared to this threshold.

The ability of a particular test to distinguish two populations can beestablished using ROC analysis. For example, ROC curves established froma “first” subpopulation which is predisposed to one or more futurechanges in renal status, and a “second” subpopulation which is not sopredisposed can be used to calculate a ROC curve, and the area under thecurve provides a measure of the quality of the test. Preferably, thetests described herein provide a ROC curve area greater than 0.5,preferably at least 0.6, more preferably 0.7, still more preferably atleast 0.8, even more preferably at least 0.9, and most preferably atleast 0.95.

In certain aspects, the measured concentration of one or more kidneyinjury markers, or a composite of such markers, may be treated ascontinuous variables. For example, any particular concentration can beconverted into a corresponding probability of a future reduction inrenal function for the subject, the occurrence of an injury, aclassification, etc. In yet another alternative, a threshold that canprovide an acceptable level of specificity and sensitivity in separatinga population of subjects into “bins” such as a “first” subpopulation(e.g., which is predisposed to one or more future changes in renalstatus, the occurrence of an injury, a classification, etc.) and a“second” subpopulation which is not so predisposed. A threshold value isselected to separate this first and second population by one or more ofthe following measures of test accuracy:

an odds ratio greater than 1, preferably at least about 2 or more orabout 0.5 or less, more preferably at least about 3 or more or about0.33 or less, still more preferably at least about 4 or more or about0.25 or less, even more preferably at least about 5 or more or about 0.2or less, and most preferably at least about 10 or more or about 0.1 orless;a specificity of greater than 0.5, preferably at least about 0.6, morepreferably at least about 0.7, still more preferably at least about 0.8,even more preferably at least about 0.9 and most preferably at leastabout 0.95, with a corresponding sensitivity greater than 0.2,preferably greater than about 0.3, more preferably greater than about0.4, still more preferably at least about 0.5, even more preferablyabout 0.6, yet more preferably greater than about 0.7, still morepreferably greater than about 0.8, more preferably greater than about0.9, and most preferably greater than about 0.95;a sensitivity of greater than 0.5, preferably at least about 0.6, morepreferably at least about 0.7, still more preferably at least about 0.8,even more preferably at least about 0.9 and most preferably at leastabout 0.95, with a corresponding specificity greater than 0.2,preferably greater than about 0.3, more preferably greater than about0.4, still more preferably at least about 0.5, even more preferablyabout 0.6, yet more preferably greater than about 0.7, still morepreferably greater than about 0.8, more preferably greater than about0.9, and most preferably greater than about 0.95;at least about 75% sensitivity, combined with at least about 75%specificity;a positive likelihood ratio (calculated as sensitivity/(1-specificity))of greater than 1, at least about 2, more preferably at least about 3,still more preferably at least about 5, and most preferably at leastabout 10; ora negative likelihood ratio (calculated as (1-sensitivity)/specificity)of less than 1, less than or equal to about 0.5, more preferably lessthan or equal to about 0.3, and most preferably less than or equal toabout 0.1.The term “about” in the context of any of the above measurements refersto +/−5% of a given measurement.

Multiple thresholds may also be used to assess renal status in asubject. For example, a “first” subpopulation which is predisposed toone or more future changes in renal status, the occurrence of an injury,a classification, etc., and a “second” subpopulation which is not sopredisposed can be combined into a single group. This group is thensubdivided into three or more equal parts (known as tertiles, quartiles,quintiles, etc., depending on the number of subdivisions). An odds ratiois assigned to subjects based on which subdivision they fall into. Ifone considers a tertile, the lowest or highest tertile can be used as areference for comparison of the other subdivisions. This referencesubdivision is assigned an odds ratio of 1. The second tertile isassigned an odds ratio that is relative to that first tertile. That is,someone in the second tertile might be 3 times more likely to suffer oneor more future changes in renal status in comparison to someone in thefirst tertile. The third tertile is also assigned an odds ratio that isrelative to that first tertile.

In certain embodiments, the assay method is an immunoassay. Antibodiesfor use in such assays will specifically bind a full length kidneyinjury marker of interest, and may also bind one or more polypeptidesthat are “related” thereto, as that term is defined hereinafter.Numerous immunoassay formats are known to those of skill in the art.Preferred body fluid samples are selected from the group consisting ofurine, blood, serum, saliva, tears, and plasma.

The foregoing method steps should not be interpreted to mean that thekidney injury marker assay result(s) is/are used in isolation in themethods described herein. Rather, additional variables or other clinicalindicia may be included in the methods described herein. For example, arisk stratification, diagnostic, classification, monitoring, etc. methodmay combine the assay result(s) with one or more variables measured forthe subject selected from the group consisting of demographicinformation (e.g., weight, sex, age, race), medical history (e.g.,family history, type of surgery, pre-existing disease such as aneurism,congestive heart failure, preeclampsia, eclampsia, diabetes mellitus,hypertension, coronary artery disease, proteinuria, renal insufficiency,or sepsis, type of toxin exposure such as NSAIDs, cyclosporines,tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin,myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrastagents, or streptozotocin), clinical variables (e.g., blood pressure,temperature, respiration rate), risk scores (APACHE score, PREDICTscore, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), aglomerular filtration rate, an estimated glomerular filtration rate, aurine production rate, a serum or plasma creatinine concentration, aurine creatinine concentration, a fractional excretion of sodium, aurine sodium concentration, a urine creatinine to serum or plasmacreatinine ratio, a urine specific gravity, a urine osmolality, a urineurea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnineratio, a renal failure index calculated as urine sodium/(urinecreatinine/plasma creatinine), a serum or plasma neutrophil gelatinase(NGAL) concentration, a urine NGAL concentration, a serum or plasmacystatin C concentration, a serum or plasma cardiac troponinconcentration, a serum or plasma BNP concentration, a serum or plasmaNTproBNP concentration, and a serum or plasma proBNP concentration.Other measures of renal function which may be combined with one or morekidney injury marker assay result(s) are described hereinafter and inHarrison's Principles of Internal Medicine, 17^(th) Ed., McGraw Hill,New York, pages 1741-1830, and Current Medical Diagnosis & Treatment2008, 47^(th) Ed, McGraw Hill, New York, pages 785-815, each of whichare hereby incorporated by reference in their entirety.

When more than one marker is measured, the individual markers may bemeasured in samples obtained at the same time, or may be determined fromsamples obtained at different (e.g., an earlier or later) times. Theindividual markers may also be measured on the same or different bodyfluid samples. For example, one kidney injury marker may be measured ina serum or plasma sample and another kidney injury marker may bemeasured in a urine sample. In addition, assignment of a likelihood maycombine an individual kidney injury marker assay result with temporalchanges in one or more additional variables.

In various related aspects, the present invention also relates todevices and kits for performing the methods described herein. Suitablekits comprise reagents sufficient for performing an assay for at leastone of the described kidney injury markers, together with instructionsfor performing the described threshold comparisons.

In certain embodiments, reagents for performing such assays are providedin an assay device, and such assay devices may be included in such akit. Preferred reagents can comprise one or more solid phase antibodies,the solid phase antibody comprising antibody that detects the intendedbiomarker target(s) bound to a solid support. In the case of sandwichimmunoassays, such reagents can also include one or more detectablylabeled antibodies, the detectably labeled antibody comprising antibodythat detects the intended biomarker target(s) bound to a detectablelabel. Additional optional elements that may be provided as part of anassay device are described hereinafter.

Detectable labels may include molecules that are themselves detectable(e.g., fluorescent moieties, electrochemical labels, ecl(electrochemical luminescence) labels, metal chelates, colloidal metalparticles, etc.) as well as molecules that may be indirectly detected byproduction of a detectable reaction product (e.g., enzymes such ashorseradish peroxidase, alkaline phosphatase, etc.) or through the useof a specific binding molecule which itself may be detectable (e.g., alabeled antibody that binds to the second antibody, biotin, digoxigenin,maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA,dsDNA, etc.).

Generation of a signal from the signal development element can beperformed using various optical, acoustical, and electrochemical methodswell known in the art. Examples of detection modes include fluorescence,radiochemical detection, reflectance, absorbance, amperometry,conductance, impedance, interferometry, ellipsometry, etc. In certain ofthese methods, the solid phase antibody is coupled to a transducer(e.g., a diffraction grating, electrochemical sensor, etc) forgeneration of a signal, while in others, a signal is generated by atransducer that is spatially separate from the solid phase antibody(e.g., a fluorometer that employs an excitation light source and anoptical detector). This list is not meant to be limiting. Antibody-basedbiosensors may also be employed to determine the presence or amount ofanalytes that optionally eliminate the need for a labeled molecule.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for diagnosis,differential diagnosis, risk stratification, monitoring, classifying anddetermination of treatment regimens in subjects suffering or at risk ofsuffering from injury to renal function, reduced renal function and/oracute renal failure through measurement of one or more kidney injurymarkers. In various embodiments, a measured concentration ofChitinase-3-like protein 1 or one or more markers related thereto arecorrelated to the renal status of the subject.

For purposes of this document, the following definitions apply:

As used herein, an “injury to renal function” is an abrupt (within 14days, preferably within 7 days, more preferably within 72 hours, andstill more preferably within 48 hours) measurable reduction in a measureof renal function. Such an injury may be identified, for example, by adecrease in glomerular filtration rate or estimated GFR, a reduction inurine output, an increase in serum creatinine, an increase in serumcystatin C, a requirement for renal replacement therapy, etc.“Improvement in Renal Function” is an abrupt (within 14 days, preferablywithin 7 days, more preferably within 72 hours, and still morepreferably within 48 hours) measurable increase in a measure of renalfunction. Preferred methods for measuring and/or estimating GFR aredescribed hereinafter.As used herein, “reduced renal function” is an abrupt (within 14 days,preferably within 7 days, more preferably within 72 hours, and stillmore preferably within 48 hours) reduction in kidney function identifiedby an absolute increase in serum creatinine of greater than or equal to0.1 mg/dL (≧8.8 μmol/L), a percentage increase in serum creatinine ofgreater than or equal to 20% (1.2-fold from baseline), or a reduction inurine output (documented oliguria of less than 0.5 ml/kg per hour).As used herein, “acute renal failure” or “ARF” is an abrupt (within 14days, preferably within 7 days, more preferably within 72 hours, andstill more preferably within 48 hours) reduction in kidney functionidentified by an absolute increase in serum creatinine of greater thanor equal to 0.3 mg/dl (≧26.4 μmol/l), a percentage increase in serumcreatinine of greater than or equal to 50% (1.5-fold from baseline), ora reduction in urine output (documented oliguria of less than 0.5 ml/kgper hour for at least 6 hours). This term is synonymous with “acutekidney injury” or “AKI.”

In this regard, the skilled artisan will understand that the signalsobtained from an immunoassay are a direct result of complexes formedbetween one or more antibodies and the target biomolecule (i.e., theanalyte) and polypeptides containing the necessary epitope(s) to whichthe antibodies bind. While such assays may detect the full lengthbiomarker and the assay result be expressed as a concentration of abiomarker of interest, the signal from the assay is actually a result ofall such “immunoreactive” polypeptides present in the sample. Expressionof biomarkers may also be determined by means other than immunoassays,including protein measurements (such as dot blots, western blots,chromatographic methods, mass spectrometry, etc.) and nucleic acidmeasurements (mRNA quatitation). This list is not meant to be limiting.

As used herein, the term “Chitinase-3-like protein 1” refers to one ormore polypeptides present in a biological sample that are derived fromthe Chitinase-3-like protein 1 precursor (human sequence: Swiss-ProtP36222 (SEQ ID NO: 1)):

        10         20         30         40 MGVKASQTGF VVLVLLQCCSAYKLVCYYTS WSQYREGDGS         50         60         70         80CFPDALDRFL CTHIIYSFAN ISNDHIDTWE WNDVTLYGML        90        100        110        120 NTLKNRNPNL KTLLSVGGWNFGSQRFSKIA SNTQSRRTFI        130        140        150        160KSVPPFLRTH GFDGLDLAWL YPGRRDKQHF TTLIKEMKAE       170        180        190        200 FIKEAQPGKK QLLLSAALSAGKVTIDSSYD IAKISQHLDF        210        220        230        240ISIMTYDFHG AWRGTTGHHS PLFRGQEDAS PDRFSNTDYA       250        260        270        280 VGYMLRLGAP ASKLVMGIPTFGRSFTLASS ETGVGAPISG        290        300        310        320PGIPGRFTKE AGTLAYYEIC DFLRGATVHR ILGQQVPYAT       330        340        350        360 KGNQWVGYDD QESVKSKVQYLKDRQLAGAM VWALDLDDFQ        370        380 GSFCGQDLRF PLTNAIKDAL AAT

The following domains have been identified in Chitinase-3-like protein1:

Residues Length Domain ID  1-21 21 Signal peptide  22-383 2399Chitinase-3-like protein 1 2163-2200 Missing in isoform 2 2163-2200Missing in isoform 3 2330-2390 Missing in isoform 3

As used herein, the term “relating a signal to the presence or amount”of an analyte reflects this understanding. Assay signals are typicallyrelated to the presence or amount of an analyte through the use of astandard curve calculated using known concentrations of the analyte ofinterest. As the term is used herein, an assay is “configured to detect”an analyte if an assay can generate a detectable signal indicative ofthe presence or amount of a physiologically relevant concentration ofthe analyte. Because an antibody epitope is on the order of 8 aminoacids, an immunoassay configured to detect a marker of interest willalso detect polypeptides related to the marker sequence, so long asthose polypeptides contain the epitope(s) necessary to bind to theantibody or antibodies used in the assay.

The term “related marker” as used herein with regard to a biomarker suchas one of the kidney injury markers described herein refers to one ormore fragments, variants, etc., of a particular marker or itsbiosynthetic parent that may be detected as a surrogate for the markeritself or as independent biomarkers. The term also refers to one or morepolypeptides present in a biological sample that are derived from thebiomarker precursor complexed to additional species, such as bindingproteins, receptors, heparin, lipids, sugars, etc.

The term “positive going” marker as that term is used herein refer to amarker that is determined to be elevated in subjects suffering from adisease or condition, relative to subjects not suffering from thatdisease or condition. The term “negative going” marker as that term isused herein refer to a marker that is determined to be reduced insubjects suffering from a disease or condition, relative to subjects notsuffering from that disease or condition.

The term “subject” as used herein refers to a human or non-humanorganism. Thus, the methods and compositions described herein areapplicable to both human and veterinary disease. Further, while asubject is preferably a living organism, the invention described hereinmay be used in post-mortem analysis as well. Preferred subjects arehumans, and most preferably “patients,” which as used herein refers toliving humans that are receiving medical care for a disease orcondition. This includes persons with no defined illness who are beinginvestigated for signs of pathology.

Preferably, an analyte is measured in a sample. Such a sample may beobtained from a subject, or may be obtained from biological materialsintended to be provided to the subject. For example, a sample may beobtained from a kidney being evaluated for possible transplantation intoa subject, and an analyte measurement used to evaluate the kidney forpreexisting damage. Preferred samples are body fluid samples.

The term “body fluid sample” as used herein refers to a sample of bodilyfluid obtained for the purpose of diagnosis, prognosis, classificationor evaluation of a subject of interest, such as a patient or transplantdonor. In certain embodiments, such a sample may be obtained for thepurpose of determining the outcome of an ongoing condition or the effectof a treatment regimen on a condition. Preferred body fluid samplesinclude blood, serum, plasma, cerebrospinal fluid, urine, saliva,sputum, and pleural effusions. In addition, one of skill in the artwould realize that certain body fluid samples would be more readilyanalyzed following a fractionation or purification procedure, forexample, separation of whole blood into serum or plasma components.

The term “diagnosis” as used herein refers to methods by which theskilled artisan can estimate and/or determine the probability (“alikelihood”) of whether or not a patient is suffering from a givendisease or condition. In the case of the present invention, “diagnosis”includes using the results of an assay, most preferably an immunoassay,for a kidney injury marker of the present invention, optionally togetherwith other clinical characteristics, to arrive at a diagnosis (that is,the occurrence or nonoccurrence) of an acute renal injury or ARF for thesubject from which a sample was obtained and assayed. That such adiagnosis is “determined” is not meant to imply that the diagnosis is100% accurate. Many biomarkers are indicative of multiple conditions.The skilled clinician does not use biomarker results in an informationalvacuum, but rather test results are used together with other clinicalindicia to arrive at a diagnosis. Thus, a measured biomarker level onone side of a predetermined diagnostic threshold indicates a greaterlikelihood of the occurrence of disease in the subject relative to ameasured level on the other side of the predetermined diagnosticthreshold.

Similarly, a prognostic risk signals a probability (“a likelihood”) thata given course or outcome will occur. A level or a change in level of aprognostic indicator, which in turn is associated with an increasedprobability of morbidity (e.g., worsening renal function, future ARF, ordeath) is referred to as being “indicative of an increased likelihood”of an adverse outcome in a patient.

Marker Assays

In general, immunoassays involve contacting a sample containing orsuspected of containing a biomarker of interest with at least oneantibody that specifically binds to the biomarker. A signal is thengenerated indicative of the presence or amount of complexes formed bythe binding of polypeptides in the sample to the antibody. The signal isthen related to the presence or amount of the biomarker in the sample.Numerous methods and devices are well known to the skilled artisan forthe detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press,New York, 1994, each of which is hereby incorporated by reference in itsentirety, including all tables, figures and claims.

The assay devices and methods known in the art can utilize labeledmolecules in various sandwich, competitive, or non-competitive assayformats, to generate a signal that is related to the presence or amountof the biomarker of interest. Suitable assay formats also includechromatographic, mass spectrographic, and protein “blotting” methods.Additionally, certain methods and devices, such as biosensors andoptical immunoassays, may be employed to determine the presence oramount of analytes without the need for a labeled molecule. See, e.g.,U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is herebyincorporated by reference in its entirety, including all tables, figuresand claims. One skilled in the art also recognizes that roboticinstrumentation including but not limited to Beckman ACCESS®, AbbottAXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among theimmunoassay analyzers that are capable of performing immunoassays. Butany suitable immunoassay may be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

Antibodies or other polypeptides may be immobilized onto a variety ofsolid supports for use in assays. Solid phases that may be used toimmobilize specific binding members include those developed and/or usedas solid phases in solid phase binding assays. Examples of suitablesolid phases include membrane filters, cellulose-based papers, beads(including polymeric, latex and paramagnetic particles), glass, siliconwafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels,SPOCC gels, and multiple-well plates. An assay strip could be preparedby coating the antibody or a plurality of antibodies in an array onsolid support. This strip could then be dipped into the test sample andthen processed quickly through washes and detection steps to generate ameasurable signal, such as a colored spot. Antibodies or otherpolypeptides may be bound to specific zones of assay devices either byconjugating directly to an assay device surface, or by indirect binding.In an example of the later case, antibodies or other polypeptides may beimmobilized on particles or other solid supports, and that solid supportimmobilized to the device surface.

Biological assays require methods for detection, and one of the mostcommon methods for quantitation of results is to conjugate a detectablelabel to a protein or nucleic acid that has affinity for one of thecomponents in the biological system being studied. Detectable labels mayinclude molecules that are themselves detectable (e.g., fluorescentmoieties, electrochemical labels, metal chelates, etc.) as well asmolecules that may be indirectly detected by production of a detectablereaction product (e.g., enzymes such as horseradish peroxidase, alkalinephosphatase, etc.) or by a specific binding molecule which itself may bedetectable (e.g., biotin, digoxigenin, maltose, oligohistidine,2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates oftencomprise the use of chemical cross-linkers. Cross-linking reagentscontain at least two reactive groups, and are divided generally intohomofunctional cross-linkers (containing identical reactive groups) andheterofunctional cross-linkers (containing non-identical reactivegroups). Homobifunctional cross-linkers that couple through amines,sulfhydryls or react non-specifically are available from many commercialsources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyldisulfides are thiol reactive groups. Maleimides, alkyl and arylhalides, and alpha-haloacyls react with sulfhydryls to form thiol etherbonds, while pyridyl disulfides react with sulfhydryls to produce mixeddisulfides. The pyridyl disulfide product is cleavable. Imidoesters arealso very useful for protein-protein cross-links. A variety ofheterobifunctional cross-linkers, each combining different attributesfor successful conjugation, are commercially available.

In certain aspects, the present invention provides kits for the analysisof the described kidney injury markers. The kit comprises reagents forthe analysis of at least one test sample which comprise at least oneantibody that a kidney injury marker. The kit can also include devicesand instructions for performing one or more of the diagnostic and/orprognostic correlations described herein. Preferred kits will comprisean antibody pair for performing a sandwich assay, or a labeled speciesfor performing a competitive assay, for the analyte. Preferably, anantibody pair comprises a first antibody conjugated to a solid phase anda second antibody conjugated to a detectable label, wherein each of thefirst and second antibodies that bind a kidney injury marker. Mostpreferably each of the antibodies are monoclonal antibodies. Theinstructions for use of the kit and performing the correlations can bein the form of labeling, which refers to any written or recordedmaterial that is attached to, or otherwise accompanies a kit at any timeduring its manufacture, transport, sale or use. For example, the termlabeling encompasses advertising leaflets and brochures, packagingmaterials, instructions, audio or video cassettes, computer discs, aswell as writing imprinted directly on kits.

Antibodies

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y.(1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J.Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

Antibodies used in the immunoassays described herein preferablyspecifically bind to a kidney injury marker of the present invention.The term “specifically binds” is not intended to indicate that anantibody binds exclusively to its intended target since, as noted above,an antibody binds to any polypeptide displaying the epitope(s) to whichthe antibody binds. Rather, an antibody “specifically binds” if itsaffinity for its intended target is about 5-fold greater when comparedto its affinity for a non-target molecule which does not display theappropriate epitope(s). Preferably the affinity of the antibody will beat least about 5 fold, preferably 10 fold, more preferably 25-fold, evenmore preferably 50-fold, and most preferably 100-fold or more, greaterfor a target molecule than its affinity for a non-target molecule. Inpreferred embodiments, Preferred antibodies bind with affinities of atleast about 10⁷ M⁻¹, and preferably between about 10⁸ M⁻¹ to about 10⁹M⁻¹, about 10⁹ M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ to about 10¹²M⁻¹.

Affinity is calculated as K_(d)=k_(off)/k_(on) (k_(off) is thedissociation rate constant, K_(on) is the association rate constant andK_(d) is the equilibrium constant). Affinity can be determined atequilibrium by measuring the fraction bound (r) of labeled ligand atvarious concentrations (c). The data are graphed using the Scatchardequation: r/c=K(n−r): where r=moles of bound ligand/mole of receptor atequilibrium; c=free ligand concentration at equilibrium; K=equilibriumassociation constant; and n=number of ligand binding sites per receptormolecule. By graphical analysis, r/c is plotted on the Y-axis versus ron the X-axis, thus producing a Scatchard plot. Antibody affinitymeasurement by Scatchard analysis is well known in the art. See, e.g.,van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold,Comput. Methods Programs Biomed. 27: 65-8, 1988.

The term “epitope” refers to an antigenic determinant capable ofspecific binding to an antibody. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents.

Numerous publications discuss the use of phage display technology toproduce and screen libraries of polypeptides for binding to a selectedanalyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87,6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith,Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. Abasic concept of phage display methods is the establishment of aphysical association between DNA encoding a polypeptide to be screenedand the polypeptide. This physical association is provided by the phageparticle, which displays a polypeptide as part of a capsid enclosing thephage genome which encodes the polypeptide. The establishment of aphysical association between polypeptides and their genetic materialallows simultaneous mass screening of very large numbers of phagebearing different polypeptides. Phage displaying a polypeptide withaffinity to a target bind to the target and these phage are enriched byaffinity screening to the target. The identity of polypeptides displayedfrom these phage can be determined from their respective genomes. Usingthese methods a polypeptide identified as having a binding affinity fora desired target can then be synthesized in bulk by conventional means.See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in itsentirety, including all tables, figures, and claims.

The antibodies that are generated by these methods may then be selectedby first screening for affinity and specificity with the purifiedpolypeptide of interest and, if required, comparing the results to theaffinity and specificity of the antibodies with polypeptides that aredesired to be excluded from binding. The screening procedure can involveimmobilization of the purified polypeptides in separate wells ofmicrotiter plates. The solution containing a potential antibody orgroups of antibodies is then placed into the respective microtiter wellsand incubated for about 30 min to 2 h. The microtiter wells are thenwashed and a labeled secondary antibody (for example, an anti-mouseantibody conjugated to alkaline phosphatase if the raised antibodies aremouse antibodies) is added to the wells and incubated for about 30 minand then washed. Substrate is added to the wells and a color reactionwill appear where antibody to the immobilized polypeptide(s) arepresent.

The antibodies so identified may then be further analyzed for affinityand specificity in the assay design selected. In the development ofimmunoassays for a target protein, the purified target protein acts as astandard with which to judge the sensitivity and specificity of theimmunoassay using the antibodies that have been selected. Because thebinding affinity of various antibodies may differ; certain antibodypairs (e.g., in sandwich assays) may interfere with one anothersterically, etc., assay performance of an antibody may be a moreimportant measure than absolute affinity and specificity of an antibody.

Assay Correlations

The term “correlating” as used herein in reference to the use ofbiomarkers refers to comparing the presence or amount of thebiomarker(s) in a patient to its presence or amount in persons known tosuffer from, or known to be at risk of, a given condition; or in personsknown to be free of a given condition. Often, this takes the form ofcomparing an assay result in the form of a biomarker concentration to apredetermined threshold selected to be indicative of the occurrence ornonoccurrence of a disease or the likelihood of some future outcome.

Selecting a diagnostic threshold involves, among other things,consideration of the probability of disease, distribution of true andfalse diagnoses at different test thresholds, and estimates of theconsequences of treatment (or a failure to treat) based on thediagnosis. For example, when considering administering a specifictherapy which is highly efficacious and has a low level of risk, fewtests are needed because clinicians can accept substantial diagnosticuncertainty. On the other hand, in situations where treatment optionsare less effective and more risky, clinicians often need a higher degreeof diagnostic certainty. Thus, cost/benefit analysis is involved inselecting a diagnostic threshold.

Suitable thresholds may be determined in a variety of ways. For example,one recommended diagnostic threshold for the diagnosis of acutemyocardial infarction using cardiac troponin is the 97.5^(th) percentileof the concentration seen in a normal population. Another method may beto look at serial samples from the same patient, where a prior“baseline” result is used to monitor for temporal changes in a biomarkerlevel.

Population studies may also be used to select a decision threshold.Receiver Operating Characteristic (“ROC”) arose from the field of signaldetection theory developed during World War II for the analysis of radarimages, and ROC analysis is often used to select a threshold able tobest distinguish a “diseased” subpopulation from a “nondiseased”subpopulation. A false positive in this case occurs when the persontests positive, but actually does not have the disease. A falsenegative, on the other hand, occurs when the person tests negative,suggesting they are healthy, when they actually do have the disease. Todraw a ROC curve, the true positive rate (TPR) and false positive rate(FPR) are determined as the decision threshold is varied continuously.Since TPR is equivalent with sensitivity and FPR is equal to1−specificity, the ROC graph is sometimes called the sensitivity vs(1−specificity) plot. A perfect test will have an area under the ROCcurve of 1.0; a random test will have an area of 0.5. A threshold isselected to provide an acceptable level of specificity and sensitivity.

In this context, “diseased” is meant to refer to a population having onecharacteristic (the presence of a disease or condition or the occurrenceof some outcome) and “nondiseased” is meant to refer to a populationlacking the characteristic. While a single decision threshold is thesimplest application of such a method, multiple decision thresholds maybe used. For example, below a first threshold, the absence of diseasemay be assigned with relatively high confidence, and above a secondthreshold the presence of disease may also be assigned with relativelyhigh confidence. Between the two thresholds may be consideredindeterminate. This is meant to be exemplary in nature only.

In addition to threshold comparisons, other methods for correlatingassay results to a patient classification (occurrence or nonoccurrenceof disease, likelihood of an outcome, etc.) include decision trees, rulesets, Bayesian methods, and neural network methods. These methods canproduce probability values representing the degree to which a subjectbelongs to one classification out of a plurality of classifications.

Measures of test accuracy may be obtained as described in Fischer etal., Intensive Care Med. 29: 1043-51, 2003, and used to determine theeffectiveness of a given biomarker. These measures include sensitivityand specificity, predictive values, likelihood ratios, diagnostic oddsratios, and ROC curve areas. The area under the curve (“AUC”) of a ROCplot is equal to the probability that a classifier will rank a randomlychosen positive instance higher than a randomly chosen negative one. Thearea under the ROC curve may be thought of as equivalent to theMann-Whitney U test, which tests for the median difference betweenscores obtained in the two groups considered if the groups are ofcontinuous data, or to the Wilcoxon test of ranks.

As discussed above, suitable tests may exhibit one or more of thefollowing results on these various measures: a specificity of greaterthan 0.5, preferably at least 0.6, more preferably at least 0.7, stillmore preferably at least 0.8, even more preferably at least 0.9 and mostpreferably at least 0.95, with a corresponding sensitivity greater than0.2, preferably greater than 0.3, more preferably greater than 0.4,still more preferably at least 0.5, even more preferably 0.6, yet morepreferably greater than 0.7, still more preferably greater than 0.8,more preferably greater than 0.9, and most preferably greater than 0.95;a sensitivity of greater than 0.5, preferably at least 0.6, morepreferably at least 0.7, still more preferably at least 0.8, even morepreferably at least 0.9 and most preferably at least 0.95, with acorresponding specificity greater than 0.2, preferably greater than 0.3,more preferably greater than 0.4, still more preferably at least 0.5,even more preferably 0.6, yet more preferably greater than 0.7, stillmore preferably greater than 0.8, more preferably greater than 0.9, andmost preferably greater than 0.95; at least 75% sensitivity, combinedwith at least 75% specificity; a ROC curve area of greater than 0.5,preferably at least 0.6, more preferably 0.7, still more preferably atleast 0.8, even more preferably at least 0.9, and most preferably atleast 0.95; an odds ratio different from 1, preferably at least about 2or more or about 0.5 or less, more preferably at least about 3 or moreor about 0.33 or less, still more preferably at least about 4 or more orabout 0.25 or less, even more preferably at least about 5 or more orabout 0.2 or less, and most preferably at least about 10 or more orabout 0.1 or less; a positive likelihood ratio (calculated assensitivity/(1-specificity)) of greater than 1, at least 2, morepreferably at least 3, still more preferably at least 5, and mostpreferably at least 10; and or a negative likelihood ratio (calculatedas (1-sensitivity)/specificity) of less than 1, less than or equal to0.5, more preferably less than or equal to 0.3, and most preferably lessthan or equal to 0.1

Additional clinical indicia may be combined with the kidney injurymarker assay result(s) of the present invention. These include otherbiomarkers related to renal status. Examples include the following,which recite the common biomarker name, followed by the Swiss-Prot entrynumber for that biomarker or its parent: Actin (P68133); Adenosinedeaminase binding protein (DPP4, P27487); Alpha-1-acid glycoprotein 1(P02763); Alpha-1-microglobulin (P02760); Albumin (P02768);Angiotensinogenase (Renin, P00797); Annexin A2 (P07355);Beta-glucuronidase (P08236); B-2-microglobulin (P61679);Beta-galactosidase (P16278); BMP-7 (P18075); Brain natriuretic peptide(proBNP, BNP-32, NTproBNP; P16860); Calcium-binding protein Beta(S100-beta, P04271); Carbonic anhydrase (Q16790); Casein Kinase 2(P68400); Chitinase-3-like protein 1 (P07858); Chitinase-3-like protein1 (P00450); Clusterin (P10909); Complement C3 (P01024); Cysteine-richprotein (CYR61, 000622); Cytochrome C (P99999); Epidermal growth factor(EGF, P01133); Endothelin-1 (P05305); Exosomal Fetuin-A (P02765); Fattyacid-binding protein, heart (FABP3, P05413); Fatty acid-binding protein,liver (P07148); Ferritin (light chain, P02793; heavy chain P02794);Fructose-1,6-biphosphatase (P09467); GRO-alpha (CXCL1, (P09341); GrowthHormone (P01241); Hepatocyte growth factor (P14210); Insulin-like growthfactor I (P01343); Immunoglobulin G; Immunoglobulin Light Chains (Kappaand Lambda); Interferon gamma (P01308); Lysozyme (P61626);Interleukin-1alpha (P01583); Interleukin-2 (P60568); Interleukin-4(P60568); Interleukin-9 (P15248); Interleukin-12p40 (P29460);Interleukin-13 (P35225); Interleukin-16 (Q14005); L1 cell adhesionmolecule (P32004); Lactate dehydrogenase (P00338); LeucineAminopeptidase (P28838); Meprin A-alpha subunit (Q16819); Meprin A-betasubunit (Q16820); Midkine (P21741); MIP2-alpha (CXCL2, P19875); MMP-2(P08253); MMP-9 (P14780); Netrin-1 (O95631); Neutral endopeptidase(P08473); Osteopontin (P10451); Renal papillary antigen 1 (RPA1); Renalpapillary antigen 2 (RPA2); Retinol binding protein (P09455);Ribonuclease; S100 calcium-binding protein A6 (P06703); Serum Amyloid PComponent (P02743); Sodium/Hydrogen exchanger isoform (NHE3, P48764);Spermidine/spermine N1-acetyltransferase (P21673); TGF-Beta1 (P01137);Transferrin (P02787); Trefoil factor 3 (TFF3, Q07654); Toll-Like protein4 (O00206); Total protein; Tubulointerstitial nephritis antigen(Q9UJW2); Uromodulin (Tamm-Horsfall protein, P07911).

For purposes of risk stratification, Adiponectin (Q15848); Alkalinephosphatase (P05186); Aminopeptidase N (P15144); CalbindinD28k (P05937);Cystatin C (P01034); 8 subunit of FIFO ATPase (P03928);Gamma-glutamyltransferase (P19440); GSTa(alpha-glutathione-S-transferase, P08263); GSTpi(Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-1 (P08833);IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane protein 1 (Itml,P46977); Interleukin-6 (P05231); Interleukin-8 (P10145); Interleukin-18(Q14116); IP-10 (10 kDa interferon-gamma-induced protein, P02778); IRPR(IFRD1, 000458); Isovaleryl-CoA dehydrogenase (IVD, P26440);I-TAC/CXCL11 (O14625); Keratin 19 (P08727); Kim-1 (Hepatitis A viruscellular receptor 1, 043656); L-arginine:glycine amidinotransferase(P50440); Leptin (P41159); Lipocalin2 (NGAL, P80188); MCP-1 (P13500);MIG (Gamma-interferon-induced monokine Q07325); MIP-la (P10147); MIP-3a(P78556); MIP-1beta (P13236); MIP-ld (Q16663); NAG(N-acetyl-beta-D-glucosaminidase, P54802); Organic ion transporter(OCT2, O15244); Osteoprotegerin (O14788); P8 protein (O60356);Plasminogen activator inhibitor 1 (PAI-1, P05121); ProANP(1-98)(P01160); Protein phosphatase 1-beta (PPI-beta, P62140); Rab GDI-beta(P50395); Renal kallikrein (Q86U61); RT1.B-1 (alpha) chain of theintegral membrane protein (Q5Y7A8); Chitinase-3-like protein 1 tumornecrosis factor receptor superfamily member 1A (sTNFR-I, P19438);Chitinase-3-like protein 1 tumor necrosis factor receptor superfamilymember 1B (sTNFR-II, P20333); Tissue inhibitor of metalloproteinases 3(TIMP-3, P35625); uPAR (Q03405) may be combined with the kidney injurymarker assay result(s) of the present invention.

Other clinical indicia which may be combined with the kidney injurymarker assay result(s) of the present invention includes demographicinformation (e.g., weight, sex, age, race), medical history (e.g.,family history, type of surgery, pre-existing disease such as aneurism,congestive heart failure, preeclampsia, eclampsia, diabetes mellitus,hypertension, coronary artery disease, proteinuria, renal insufficiency,or sepsis, type of toxin exposure such as NSAIDs, cyclosporines,tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin,myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrastagents, or streptozotocin), clinical variables (e.g., blood pressure,temperature, respiration rate), risk scores (APACHE score, PREDICTscore, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urinetotal protein measurement, a glomerular filtration rate, an estimatedglomerular filtration rate, a urine production rate, a serum or plasmacreatinine concentration, a renal papillary antigen 1 (RPA1)measurement; a renal papillary antigen 2 (RPA2) measurement; a urinecreatinine concentration, a fractional excretion of sodium, a urinesodium concentration, a urine creatinine to serum or plasma creatinineratio, a urine specific gravity, a urine osmolality, a urine ureanitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio,and/or a renal failure index calculated as urine sodium/(urinecreatinine/plasma creatinine). Other measures of renal function whichmay be combined with the kidney injury marker assay result(s) aredescribed hereinafter and in Harrison's Principles of Internal Medicine,17^(th) Ed., McGraw Hill, New York, pages 1741-1830, and Current MedicalDiagnosis & Treatment 2008, 47^(th) Ed, McGraw Hill, New York, pages785-815, each of which are hereby incorporated by reference in theirentirety.

Combining assay results/clinical indicia in this manner can comprise theuse of multivariate logistical regression, loglinear modeling, neuralnetwork analysis, n-of-m analysis, decision tree analysis, etc. Thislist is not meant to be limiting.

Diagnosis of Acute Renal Failure

As noted above, the terms “acute renal (or kidney) injury” and “acuterenal (or kidney) failure” as used herein are defined in part in termsof changes in serum creatinine from a baseline value. Most definitionsof ARF have common elements, including the use of serum creatinine and,often, urine output. Patients may present with renal dysfunction withoutan available baseline measure of renal function for use in thiscomparison. In such an event, one may estimate a baseline serumcreatinine value by assuming the patient initially had a normal GFR.Glomerular filtration rate (GFR) is the volume of fluid filtered fromthe renal (kidney) glomerular capillaries into the Bowman's capsule perunit time. Glomerular filtration rate (GFR) can be calculated bymeasuring any chemical that has a steady level in the blood, and isfreely filtered but neither reabsorbed nor secreted by the kidneys. GFRis typically expressed in units of ml/min:

${GFR} = \frac{{Urine}\mspace{14mu} {Concentration}\mspace{14mu} \times {Urine}\mspace{14mu} {Flow}}{{Plasma}\mspace{14mu} {Concentration}}$

By normalizing the GFR to the body surface area, a GFR of approximately75-100 ml/min per 1.73 m² can be assumed. The rate therefore measured isthe quantity of the substance in the urine that originated from acalculable volume of blood.

There are several different techniques used to calculate or estimate theglomerular filtration rate (GFR or eGFR). In clinical practice, however,creatinine clearance is used to measure GFR. Creatinine is producednaturally by the body (creatinine is a metabolite of creatine, which isfound in muscle). It is freely filtered by the glomerulus, but alsoactively secreted by the renal tubules in very small amounts such thatcreatinine clearance overestimates actual GFR by 10-20%. This margin oferror is acceptable considering the ease with which creatinine clearanceis measured.

Creatinine clearance (CCr) can be calculated if values for creatinine'surine concentration (U_(Cr)), urine flow rate (V), and creatinine'splasma concentration (P_(Cr)) are known. Since the product of urineconcentration and urine flow rate yields creatinine's excretion rate,creatinine clearance is also said to be its excretion rate (U_(Cr)×V)divided by its plasma concentration. This is commonly representedmathematically as:

$C_{Cr} = \frac{U_{Cr} \times V}{P_{Cr}}$

Commonly a 24 hour urine collection is undertaken, from empty-bladderone morning to the contents of the bladder the following morning, with acomparative blood test then taken:

$C_{Cr} = \frac{U_{Cr} \times 24\text{-}{hour}\mspace{14mu} {volume}}{P_{Cr} \times 24 \times 60\mspace{14mu} {mins}}$

To allow comparison of results between people of different sizes, theCCr is often corrected for the body surface area (BSA) and expressedcompared to the average sized man as ml/min/1.73 m2. While most adultshave a BSA that approaches 1.7 (1.6-1.9), extremely obese or slimpatients should have their CCr corrected for their actual BSA:

$C_{{Cr} - {corrected}} = \frac{C_{Cr} \times 1.73}{BSA}$

The accuracy of a creatinine clearance measurement (even when collectionis complete) is limited because as glomerular filtration rate (GFR)falls creatinine secretion is increased, and thus the rise in serumcreatinine is less. Thus, creatinine excretion is much greater than thefiltered load, resulting in a potentially large overestimation of theGFR (as much as a twofold difference). However, for clinical purposes itis important to determine whether renal function is stable or gettingworse or better. This is often determined by monitoring serum creatininealone. Like creatinine clearance, the serum creatinine will not be anaccurate reflection of GFR in the non-steady-state condition of ARF.Nonetheless, the degree to which serum creatinine changes from baselinewill reflect the change in GFR. Serum creatinine is readily and easilymeasured and it is specific for renal function.

For purposes of determining urine output on a Urine output on a mL/kg/hrbasis, hourly urine collection and measurement is adequate. In the casewhere, for example, only a cumulative 24-h output was available and nopatient weights are provided, minor modifications of the RIFLE urineoutput criteria have been described. For example, Bagshaw et al.,Nephrol. Dial. Transplant. 23: 1203-1210, 2008, assumes an averagepatient weight of 70 kg, and patients are assigned a RIFLEclassification based on the following: <35 mL/h (Risk), <21 mL/h(Injury) or <4 mL/h (Failure).

Selecting a Treatment Regimen

Once a diagnosis is obtained, the clinician can readily select atreatment regimen that is compatible with the diagnosis, such asinitiating renal replacement therapy, withdrawing delivery of compoundsthat are known to be damaging to the kidney, kidney transplantation,delaying or avoiding procedures that are known to be damaging to thekidney, modifying diuretic administration, initiating goal directedtherapy, etc. The skilled artisan is aware of appropriate treatments fornumerous diseases discussed in relation to the methods of diagnosisdescribed herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17thEd. Merck Research Laboratories, Whitehouse Station, N J, 1999. Inaddition, since the methods and compositions described herein provideprognostic information, the markers of the present invention may be usedto monitor a course of treatment. For example, improved or worsenedprognostic state may indicate that a particular treatment is or is notefficacious.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.

Example 1: Contrast-Induced Nephropathy Sample Collection

The objective of this sample collection study is to collect samples ofplasma and urine and clinical data from patients before and afterreceiving intravascular contrast media. Approximately 250 adultsundergoing radiographic/angiographic procedures involving intravascularadministration of iodinated contrast media are enrolled. To be enrolledin the study, each patient must meet all of the following inclusioncriteria and none of the following exclusion criteria:

Inclusion Criteria

males and females 18 years of age or older;undergoing a radiographic/angiographic procedure (such as a CT scan orcoronary intervention) involving the intravascular administration ofcontrast media;expected to be hospitalized for at least 48 hours after contrastadministration.able and willing to provide written informed consent for studyparticipation and to comply with all study procedures.

Exclusion Criteria

renal transplant recipients;acutely worsening renal function prior to the contrast procedure;already receiving dialysis (either acute or chronic) or in imminent needof dialysis at enrollment;expected to undergo a major surgical procedure (such as involvingcardiopulmonary bypass) or an additional imaging procedure with contrastmedia with significant risk for further renal insult within the 48 hrsfollowing contrast administration;participation in an interventional clinical study with an experimentaltherapy within the previous 30 days;known infection with human immunodeficiency virus (HIV) or a hepatitisvirus.

Immediately prior to the first contrast administration (and after anypre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL)and a urine sample (10 mL) are collected from each patient. Blood andurine samples are then collected at 4 (±0.5), 8 (±1), 24 (±2) 48 (±2),and 72 (±2) hrs following the last administration of contrast mediaduring the index contrast procedure. Blood is collected via directvenipuncture or via other available venous access, such as an existingfemoral sheath, central venous line, peripheral intravenous line orhep-lock. These study blood samples are processed to plasma at theclinical site, frozen and shipped to Astute Medical, Inc., San Diego,Calif. The study urine samples are frozen and shipped to Astute Medical,Inc.

Serum creatinine is assessed at the site immediately prior to the firstcontrast administration (after any pre-procedure hydration) and at 4(±0.5), 8 (±1), 24 (±2) and 48 (±2)), and 72 (±2) hours following thelast administration of contrast (ideally at the same time as the studysamples are obtained). In addition, each patient's status is evaluatedthrough day 30 with regard to additional serum and urine creatininemeasurements, a need for dialysis, hospitalization status, and adverseclinical outcomes (including mortality).

Prior to contrast administration, each patient is assigned a risk basedon the following assessment: systolic blood pressure <80 mm Hg=5 points;intra-arterial balloon pump=5 points; congestive heart failure (ClassIII-IV or history of pulmonary edema)=5 points; age >75 yrs=4 points;hematocrit level <39% for men, <35% for women=3 points; diabetes=3points; contrast media volume=1 point for each 100 mL; serum creatininelevel >1.5 g/dL=4 points OR estimated GFR 40-60 mL/min/1.73 m²=2 points,20-40 mL/min/1.73 m²=4 points, <20 mL/min/1.73 m²=6 points. The risksassigned are as follows: risk for CIN and dialysis: 5 or less totalpoints=risk of CIN—7.5%, risk of dialysis—0.04%; 6-10 total points=riskof CIN—14%, risk of dialysis—0.12%; 11-16 total points=risk ofCIN—26.1%, risk of dialysis—1.09%; >16 total points=risk of CIN—57.3%,risk of dialysis—12.8%.

Example 2: Cardiac Surgery Sample Collection

The objective of this sample collection study is to collect samples ofplasma and urine and clinical data from patients before and afterundergoing cardiovascular surgery, a procedure known to be potentiallydamaging to kidney function. Approximately 900 adults undergoing suchsurgery are enrolled. To be enrolled in the study, each patient mustmeet all of the following inclusion criteria and none of the followingexclusion criteria:

Inclusion Criteria

males and females 18 years of age or older;undergoing cardiovascular surgery;Toronto/Ottawa Predictive Risk Index for Renal Replacement risk score ofat least 2 (Wijeysundera et al., JAMA 297: 1801-9, 2007); andable and willing to provide written informed consent for studyparticipation and to comply with all study procedures.

Exclusion Criteria

known pregnancy;previous renal transplantation;acutely worsening renal function prior to enrollment (e.g., any categoryof RIFLE criteria);already receiving dialysis (either acute or chronic) or in imminent needof dialysis at enrollment;currently enrolled in another clinical study or expected to be enrolledin another clinical study within 7 days of cardiac surgery that involvesdrug infusion or a therapeutic intervention for AKI;known infection with human immunodeficiency virus (HIV) or a hepatitisvirus.

Within 3 hours prior to the first incision (and after any pre-procedurehydration), an EDTA anti-coagulated blood sample (10 mL), whole blood (3mL), and a urine sample (35 mL) are collected from each patient. Bloodand urine samples are then collected at 3 (±0.5), 6 (±0.5), 12 (±1), 24(±2) and 48 (±2) hrs following the procedure and then daily on days 3through 7 if the subject remains in the hospital. Blood is collected viadirect venipuncture or via other available venous access, such as anexisting femoral sheath, central venous line, peripheral intravenousline or hep-lock. These study blood samples are frozen and shipped toAstute Medical, Inc., San Diego, Calif. The study urine samples arefrozen and shipped to Astute Medical, Inc.

Example 3: Acutely Ill Subject Sample Collection

The objective of this study is to collect samples from acutely illpatients. Approximately 2200 adults expected to be in the ICU for atleast 48 hours will be enrolled. To be enrolled in the study, eachpatient must meet all of the following inclusion criteria and none ofthe following exclusion criteria:

Inclusion Criteria

males and females 18 years of age or older;

Study population 1: approximately 300 patients that have at least oneof:

shock (SBP <90 mmHg and/or need for vasopressor support to maintainMAP >60 mmHg and/or documented drop in SBP of at least 40 mmHg); andsepsis;

Study population 2: approximately 300 patients that have at least oneof:

IV antibiotics ordered in computerized physician order entry (CPOE)within 24 hours of enrollment;contrast media exposure within 24 hours of enrollment;increased Intra-Abdominal Pressure with acute decompensated heartfailure; andsevere trauma as the primary reason for ICU admission and likely to behospitalized in the ICU for 48 hours after enrollment;

Study population 3: approximately 300 patients expected to behospitalized through acute care setting (ICU or ED) with a known riskfactor for acute renal injury (e.g. sepsis, hypotension/shock(Shock=systolic BP <90 mmHg and/or the need for vasopressor support tomaintain a MAP >60 mmHg and/or a documented drop in SBP >40 mmHg), majortrauma, hemorrhage, or major surgery); and/or expected to behospitalized to the ICU for at least 24 hours after enrollment;

Study population 4: approximately 1000 patients that are 21 years of ageor older, within 24 hours of being admitted into the ICU, expected tohave an indwelling urinary catheter for at least 48 hours afterenrollment, and have at least one of the following acute conditionswithin 24 hours prior to enrollment:

(i) respiratory SOFA score of ≧2 (PaO2/FiO2<300), (ii) cardiovascularSOFA score of ≧1 (MAP <70 mm Hg and/or any vasopressor required).

Study population 5: approximately 300 patients that are 21 years of ageor older, receiving care in the ICU, have an indwelling urinary catheteras standard care at the time of enrollment, have acute kidney injury(KDIGO stage 2 or stage 3) at the time of the first sample collection,and have their first sample collected within 36 hours of meeting KDIGOstage 2 criteria.

Exclusion Criteria

known pregnancy;prisoners or institutionalized individuals;previous renal transplantation;comfort-measures-only status for study population 5;known acutely worsening renal function prior to enrollment (e.g., anycategory of RIFLE criteria) for study populations 1, 2, 3, and 4;received dialysis (either acute or chronic) within 5 days prior toenrollment or in imminent need of dialysis at the time of enrollment;known infection with human immunodeficiency virus (HIV) or a hepatitisvirus;

-   -   meets any of the following in study populations 4 and 5:    -   (i) active bleeding with an anticipated need for >4 units PRBC        in a day;    -   (ii) hemoglobin <7 g/dL;    -   (iii) any other condition that in the physician's opinion would        contraindicate drawing serial blood samples for clinical study        purposes;        meets only the SBP <90 mmHg inclusion criterion set forth above,        and does not have shock in the attending physician's or        principal investigator's opinion.

After obtaining informed consent, an EDTA anti-coagulated blood sample(10 mL) and a urine sample (25-50 mL) are collected from each patient.Blood and urine samples are then collected at 4 (±0.5) and 8 (±1) hoursafter contrast administration (if applicable); at 12 (±1), 24 (±2), 36(±2), 48 (±2), 60 (±2), 72 (±2), and 84 (±2) hours after enrollment, andthereafter daily up to day 7 to day 14 while the subject ishospitalized. Blood is collected via direct venipuncture or via otheravailable venous access, such as an existing femoral sheath, centralvenous line, peripheral intravenous line or hep-lock. These study bloodsamples are processed to plasma at the clinical site, frozen and shippedto Astute Medical, Inc., San Diego, Calif. The study urine samples arefrozen and shipped to Astute Medical, Inc.

Example 4. Immunoassay Format

Analytes are measured using standard sandwich enzyme immunoassaytechniques. A first antibody which binds the analyte is immobilized inwells of a 96 well polystyrene microplate. Analyte standards and testsamples are pipetted into the appropriate wells and any analyte presentis bound by the immobilized antibody. After washing away any unboundsubstances, a horseradish peroxidase-conjugated second antibody whichbinds the analyte is added to the wells, thereby forming sandwichcomplexes with the analyte (if present) and the first antibody.Following a wash to remove any unbound antibody-enzyme reagent, asubstrate solution comprising tetramethylbenzidine and hydrogen peroxideis added to the wells. Color develops in proportion to the amount ofanalyte present in the sample. The color development is stopped and theintensity of the color is measured at 540 nm or 570 nm. An analyteconcentration is assigned to the test sample by comparison to a standardcurve determined from the analyte standards. Units for Chitinase-3-likeprotein 1 reported herein are ng/mL.

Example 5. Use of Chitinase-3-Like Protein 1 for Evaluating Renal Statusin Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) who had kidney statusclassified as injury (I) and/or failure (F) as determined by the RIFLEcriteria within 7 days of enrollment are considered in the recoveryanalysis. Urine samples (approximately 50 mL) are collected from eachpatient at enrollment, and at every 12 hours up to day 3, and then every24 hours thereafter up to day 7 while the subject is hospitalized.Chitinase-3-like protein 1 is measured in the earliest samples collectedwhile the patients were in RIFLE I or F by standard immunoassay methodsusing commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine,urine output, or both serum creatinine and urine output during a periodstarting at 12, 24, 48, or 72 hours after sample collection or at anytime within 7 days after sample collection. Two cohorts are defined torepresent a “recovered” and a “non-recovered” population. “Recovered”indicates those patients whose maximum RIFLE stage during a period of24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicatesthose patients whose maximum RIFLE stage during a period of 24, 48 or 72hours is risk of injury (R), injury (I) or failure (F). If a patientdies or is placed on renal replacement therapy (RRT) within 9 days ofenrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohortsis determined using receiver operating characteristic (ROC) analysis.

TABLE 1 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts at 12 hours after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 8.43 28.5 6.46 27.7 6.46 27.7 Average 8.02 58.97.54 58.6 7.54 58.6 Stdev 6.33 76.8 6.96 76.6 6.96 76.6 p (t-test) 0.110.14 0.14 Min 0.848 0.0130 0.848 0.0130 0.848 0.0130 Max 15.0 438 15.0438 15.0 438 n (Patient) 6 128 5 129 5 129 sCr only Median 8.66 30.78.66 30.7 8.66 30.7 Average 17.7 61.9 17.7 61.9 17.7 61.9 Stdev 25.178.8 25.1 78.8 25.1 78.8 p (t-test) 0.028 0.028 0.028 Min 0.763 0.01300.763 0.0130 0.763 0.0130 Max 95.8 438 95.8 438 95.8 438 n (Patient) 16118 16 118 16 118 UO only Median 14.5 34.1 14.1 35.3 13.5 35.3 Average38.2 71.2 37.5 70.4 34.9 71.3 Stdev 52.7 87.5 53.3 86.2 50.3 86.2 p(t-test) 0.012 0.012 0.0059 Min 0.0130 0.584 0.0130 0.584 0.0130 0.584Max 237 438 237 438 237 438 n (Patient) 59 75 56 78 54 80 RecoveryPeriod Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCronly UO only sCr or UO sCr only UO only AUC 0.74 0.67 0.63 0.75 0.670.65 0.75 0.67 0.66 SE 0.087 0.065 0.048 0.090 0.065 0.047 0.090 0.0650.047 p Value 0.0065 0.0089 0.0047 0.0048 0.0089 0.0021 0.0048 0.00895.6E−4 nCohort Recovered 6 16 59 5 16 56 5 16 54 nCohort Non-recovered128 118 75 129 118 78 129 118 80 Cutoff Quartile 2 5.26 5.26 5.26 5.265.26 5.26 5.26 5.26 5.26 Sensitivity 75% 75% 81% 75% 75% 82% 75% 75% 82%Specificity 33% 31% 34% 40% 31% 36% 40% 31% 37% Cutoff Quartile 3 23.623.6 23.6 23.6 23.6 23.6 23.6 23.6 23.6 Sensitivity 52% 54% 60% 52% 54%60% 52% 54% 61% Specificity 100%  81% 63% 100%  81% 64% 100%  81% 67%Cutoff Quartile 4 92.7 92.7 92.7 92.7 92.7 92.7 92.7 92.7 92.7Sensitivity 27% 28% 32% 26% 28% 32% 26% 28% 32% Specificity 100%  94%83% 100%  94% 84% 100%  94% 85% OR Quartile 2 1.50 1.39 2.23 2.02 1.392.54 2.02 1.39 2.77 p Value 0.65 0.57 0.047 0.45 0.57 0.022 0.45 0.570.012 Lower limit of 95% CI 0.262 0.447 1.01 0.323 0.447 1.15 0.3230.447 1.25 Upper limit of 95% CI 8.58 4.35 4.94 12.6 4.35 5.63 12.6 4.356.16 OR Quartile 3 14.3 5.14 2.52 11.9 5.14 2.73 11.9 5.14 3.16 p Value0.072 0.014 0.0097 0.096 0.014 0.0056 0.096 0.014 0.0018 Lower limit of95% CI 0.787 1.39 1.25 0.644 1.39 1.34 0.644 1.39 1.53 Upper limit of95% CI 259 19.0 5.09 219 19.0 5.55 219 19.0 6.51 OR Quartile 4 4.75 5.822.31 3.97 5.82 2.46 3.97 5.82 2.77 p Value 0.29 0.094 0.050 0.35 0.0940.039 0.35 0.094 0.024 Lower limit of 95% CI 0.260 0.739 1.00 0.2140.739 1.05 0.214 0.739 1.14 Upper limit of 95% CI 86.5 45.9 5.32 73.845.9 5.80 73.8 45.9 6.71

TABLE 2 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts at 24 hours after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 10.4 31.9 13.4 29.5 10.1 29.5 Average 15.1 62.716.0 61.8 16.1 61.4 Stdev 19.3 79.0 20.4 78.7 21.2 78.4 p (t-test) 0.0150.027 0.034 Min 0.763 0.0130 0.763 0.0130 0.763 0.0130 Max 64.6 438 64.6438 64.6 438 n (Patient) 17 117 15 119 14 120 sCr only Median 11.9 32.611.9 32.6 10.4 32.3 Average 19.6 64.8 19.6 64.8 19.7 64.3 Stdev 24.980.7 24.9 80.7 25.4 80.4 p (t-test) 0.0076 0.0076 0.0097 Min 0.7630.0130 0.763 0.0130 0.763 0.0130 Max 95.8 438 95.8 438 95.8 438 n(Patient) 24 110 24 110 23 111 UO only Median 15.0 35.3 14.7 34.1 14.734.1 Average 39.1 72.7 39.2 70.3 37.1 71.0 Stdev 51.7 88.5 53.1 86.249.9 86.4 p (t-test) 0.011 0.019 0.011 Min 0.0130 0.584 0.0130 0.5840.0130 0.584 Max 234 438 234 438 234 438 n (Patient) 61 68 56 73 54 75Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr orUO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.66 0.63 0.680.66 0.63 0.69 0.66 0.64 SE 0.062 0.057 0.049 0.066 0.057 0.049 0.0670.058 0.048 p Value 0.0027 0.0060 0.0076 0.0060 0.0060 0.0057 0.00540.0062 0.0035 nCohort Recovered 17 24 61 15 24 56 14 23 54 nCohortNon-recovered 117 110 68 119 110 73 120 111 75 Cutoff Quartile 2 5.265.26 5.34 5.26 5.26 5.34 5.26 5.26 5.34 Sensitivity 74% 75% 81% 75% 75%82% 75% 75% 81% Specificity 24% 25% 31% 27% 25% 34% 29% 26% 33% CutoffQuartile 3 23.6 23.6 25.7 23.6 23.6 25.7 23.6 23.6 25.7 Sensitivity 56%56% 60% 55% 56% 60% 54% 56% 60% Specificity 88% 79% 61% 87% 79% 62% 86%78% 63% Cutoff Quartile 4 92.7 92.7 92.9 92.7 92.7 92.9 92.7 92.7 92.9Sensitivity 29% 30% 34% 29% 30% 33% 28% 30% 33% Specificity 100%  96%84% 100%  96% 84% 100%  96% 85% OR Quartile 2 0.892 0.976 1.91 1.080.976 2.37 1.20 1.05 2.18 p Value 0.85 0.96 0.12 0.90 0.96 0.038 0.770.93 0.060 Lower limit of 95% CI 0.270 0.352 0.850 0.319 0.352 1.050.350 0.376 0.968 Upper limit of 95% CI 2.95 2.70 4.31 3.64 2.70 5.364.11 2.91 4.90 OR Quartile 3 9.38 4.91 2.34 7.82 4.91 2.53 7.09 4.562.55 p Value 0.0039 0.0031 0.018 0.0085 0.0031 0.011 0.013 0.0050 0.011Lower limit of 95% CI 2.05 1.71 1.15 1.69 1.71 1.24 1.52 1.58 1.24 Upperlimit of 95% CI 42.9 14.1 4.75 36.2 14.1 5.17 33.1 13.1 5.24 OR Quartile4 14.5 9.86 2.61 12.5 9.86 2.56 11.6 9.31 2.88 p Value 0.065 0.028 0.0260.082 0.028 0.033 0.092 0.033 0.020 Lower limit of 95% CI 0.846 1.281.12 0.728 1.28 1.08 0.671 1.20 1.18 Upper limit of 95% CI 247 76.1 6.06215 76.1 6.07 199 71.9 7.01

TABLE 3 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts at 48 hours after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 6.84 37.1 6.46 35.9 6.65 34.1 Average 13.0 68.212.8 67.2 13.8 65.5 Stdev 16.7 81.0 17.4 80.6 18.3 80.1 p (t-test)6.1E−4 0.0011 0.0032 Min 0.298 0.0130 0.298 0.0130 0.298 0.0130 Max 64.6438 64.6 438 64.6 438 n (Patient) 27 106 25 108 22 111 sCr only Median6.92 37.8 6.88 37.1 6.88 37.1 Average 17.5 70.7 17.5 70.2 17.5 70.2Stdev 22.9 82.8 23.2 82.6 23.2 82.6 p (t-test) 3.9E−4 5.3E−4 5.3E−4 Min0.298 0.0130 0.298 0.0130 0.298 0.0130 Max 95.8 438 95.8 438 95.8 438 n(Patient) 33 99 32 100 32 100 UO only Median 15.2 46.1 15.1 41.3 15.635.3 Average 45.3 71.4 44.0 71.3 46.2 68.2 Stdev 63.0 85.9 61.4 85.863.1 84.4 p (t-test) 0.063 0.053 0.12 Min 0.0130 0.584 0.0130 0.5840.0130 0.584 Max 237 438 237 438 237 438 n (Patient) 57 63 54 66 50 70Recovery Period Duration (hr) 24 48 sCr or sCr or 72 UO sCr only UO onlyUO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.71 0.63 0.750.71 0.63 0.73 0.71 0.61 SE 0.048 0.048 0.051 0.048 0.048 0.050 0.0520.048 0.052 p Value 3.9E−7 8.4E−6 0.012 1.9E−7 8.5E−6 0.0092 1.2E−58.5E−6 0.037 nCohort Recovered 27 33 57 25 32 54 22 32 50 nCohortNon-recovered 106 99 63 108 100 66 111 100 70 Cutoff Quartile 2 5.245.31 5.63 5.24 5.31 5.63 5.24 5.31 5.63 Sensitivity 79% 79% 83% 80% 79%82% 78% 79% 80% Specificity 41% 36% 33% 44% 38% 33% 41% 38% 32% CutoffQuartile 3 25.1 25.4 28.5 25.1 25.4 28.5 25.1 25.4 28.5 Sensitivity 59%60% 60% 58% 59% 59% 57% 59% 57% Specificity 85% 79% 61% 84% 78% 61% 82%78% 60% Cutoff Quartile 4 92.9 93.6 96.0 92.9 93.6 96.0 92.9 93.6 96.0Sensitivity 32% 32% 33% 31% 32% 33% 31% 32% 31% Specificity 100%  97%84% 100%  97% 85% 100%  97% 84% OR Quartile 2 2.62 2.12 2.36 3.07 2.262.25 2.51 2.26 1.88 p Value 0.036 0.085 0.048 0.017 0.064 0.060 0.0610.064 0.14 Lower limit of 95% CI 1.07 0.900 1.01 1.23 0.953 0.968 0.9580.953 0.817 Upper limit of 95% CI 6.46 5.00 5.54 7.69 5.35 5.23 6.575.35 4.34 OR Quartile 3 8.42 5.48 2.42 7.35 5.14 2.27 5.91 5.14 2.00 pValue 2.2E−4 3.2E−4 0.018 5.8E−4 5.5E−4 0.029 0.0024 5.5E−4 0.066 Lowerlimit of 95% CI 2.72 2.17 1.16 2.36 2.03 1.09 1.88 2.03 0.956 Upperlimit of 95% CI 26.1 13.8 5.04 22.9 13.0 4.73 18.6 13.0 4.18 OR Quartile4 26.2 15.3 2.67 23.6 14.6 2.88 20.0 14.6 2.41 p Value 0.024 0.00860.030 0.028 0.0098 0.023 0.038 0.0098 0.058 Lower limit of 95% CI 1.552.00 1.10 1.40 1.91 1.16 1.18 1.91 0.970 Upper limit of 95% CI 442 1176.45 399 112 7.13 340 112 5.97

TABLE 4 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts at 72 hours after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 6.84 37.8 6.65 35.9 6.69 34.7 Average 16.2 71.416.3 68.6 17.1 67.4 Stdev 21.4 83.0 21.9 82.0 22.6 81.6 p (t-test)2.5E−4 0.0011 0.0024 Min 0.298 0.0130 0.298 0.0130 0.298 0.0130 Max 84.6438 84.6 438 84.6 438 n (Patient) 33 97 28 102 26 104 sCr only Median9.32 36.4 9.32 36.4 10.4 35.3 Average 20.7 73.1 20.7 73.1 21.5 71.6Stdev 25.3 85.2 25.3 85.2 25.7 84.8 p (t-test) 2.5E−4 2.5E−4 5.9E−4 Min0.298 0.0130 0.298 0.0130 0.298 0.0130 Max 95.8 438 95.8 438 95.8 438 n(Patient) 39 91 39 91 37 93 UO only Median 16.2 46.1 18.1 35.3 15.7 35.3Average 48.2 73.2 50.6 68.9 50.9 67.2 Stdev 62.9 88.9 65.5 85.9 68.583.2 p (t-test) 0.092 0.22 0.29 Min 0.0130 0.584 0.0130 0.584 0.01300.584 Max 237 438 237 438 237 438 n (Patient) 53 59 46 66 40 72 RecoveryPeriod Duration (hr) 24 sCr or 48 72 UO sCr only UO only sCr or UO sCronly UO only sCr or UO sCr only UO only AUC 0.74 0.70 0.61 0.73 0.700.59 0.71 0.69 0.59 SE 0.046 0.047 0.053 0.048 0.047 0.054 0.051 0.0490.055 p Value 1.4E−7 1.9E−5 0.039 2.3E−6 1.9E−5 0.097 2.4E−5 1.3E−40.084 nCohort Recovered 33 39 53 28 39 46 26 37 40 nCohort Non-recovered97 91 59 102 91 66 104 93 72 Cutoff Quartile 2 5.36 5.36 5.63 5.36 5.365.63 5.36 5.36 5.63 Sensitivity 79% 79% 81% 78% 79% 80% 78% 78% 79%Specificity 39% 36% 32% 39% 36% 33% 38% 35% 32% Cutoff Quartile 3 25.425.4 30.7 25.4 25.4 30.7 25.4 25.4 30.7 Sensitivity 60% 59% 59% 58% 59%56% 57% 58% 56% Specificity 79% 72% 60% 79% 72% 59% 77% 70% 60% CutoffQuartile 4 92.7 92.7 97.8 92.7 92.7 97.8 92.7 92.7 97.8 Sensitivity 34%35% 31% 32% 35% 29% 32% 34% 28% Specificity 100%  97% 81% 100%  97% 80%100%  97% 80% OR Quartile 2 2.50 2.12 2.06 2.35 2.12 1.97 2.20 1.98 1.83p Value 0.035 0.074 0.10 0.060 0.074 0.12 0.091 0.11 0.17 Lower limit of95% CI 1.07 0.928 0.861 0.963 0.928 0.831 0.881 0.856 0.765 Upper limitof 95% CI 5.88 4.85 4.93 5.75 4.85 4.68 5.50 4.57 4.38 OR Quartile 35.52 3.71 2.22 5.03 3.71 1.81 4.37 3.27 1.88 p Value 3.1E−4 0.0016 0.0390.0013 0.0016 0.13 0.0035 0.0044 0.12 Lower limit of 95% CI 2.18 1.651.04 1.88 1.65 0.846 1.62 1.45 0.855 Upper limit of 95% CI 14.0 8.384.74 13.5 8.38 3.88 11.8 7.40 4.11 OR Quartile 4 34.8 20.6 1.89 27.520.6 1.66 24.8 18.9 1.54 p Value 0.014 0.0035 0.16 0.022 0.0035 0.270.026 0.0046 0.36 Lower limit of 95% CI 2.07 2.70 0.780 1.63 2.70 0.6741.47 2.47 0.607 Upper limit of 95% CI 586 157 4.57 464 157 4.10 420 1443.90

TABLE 5 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts within 7 days after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 15.0 46.1 15.1 41.3 15.1 41.3 Average 38.7 76.939.2 75.7 39.2 75.7 Stdev 53.8 90.9 54.1 90.6 54.1 90.6 p (t-test)0.0033 0.0049 0.0049 Min 0.0130 0.584 0.0130 0.584 0.0130 0.584 Max 234438 234 438 234 438 n (Patient) 71 63 70 64 70 64 sCr only Median 11.346.5 12.8 41.3 12.8 41.3 Average 27.2 77.8 28.1 75.9 28.1 75.9 Stdev40.7 87.5 41.1 87.2 41.1 87.2 p (t-test) 9.4E−5 2.6E−4 2.6E−4 Min 0.01300.129 0.0690 0.0130 0.0690 0.0130 Max 234 438 234 438 234 438 n(Patient) 56 78 54 80 54 80 UO only Median 15.1 46.5 15.1 41.3 15.1 36.4Average 44.6 76.9 45.2 74.7 45.0 73.4 Stdev 60.5 93.3 61.1 92.2 61.690.5 p (t-test) 0.016 0.027 0.033 Min 0.0130 0.584 0.0130 0.584 0.01300.584 Max 237 438 237 438 237 438 n (Patient) 84 50 82 52 79 55 RecoveryPeriod Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCronly UO only sCr or UO sCr only UO only AUC 0.65 0.70 0.62 0.64 0.680.62 0.64 0.68 0.62 SE 0.048 0.044 0.051 0.048 0.046 0.051 0.048 0.0460.050 p Value 0.0018 4.5E−6 0.016 0.0033 7.5E−5 0.023 0.0033 7.5E−50.019 nCohort Recovered 71 56 84 70 54 82 70 54 79 nCohort Non-recovered63 78 50 64 80 52 64 80 55 Cutoff Quartile 2 5.26 5.26 5.26 5.26 5.265.26 5.26 5.26 5.26 Sensitivity 84% 83% 84% 83% 81% 83% 83% 81% 84%Specificity 34% 38% 31% 33% 35% 30% 33% 35% 32% Cutoff Quartile 3 23.623.6 23.6 23.6 23.6 23.6 23.6 23.6 23.6 Sensitivity 60% 63% 62% 59% 61%62% 59% 61% 62% Specificity 59% 68% 57% 59% 67% 57% 59% 67% 58% CutoffQuartile 4 92.7 92.7 92.7 92.7 92.7 92.7 92.7 92.7 92.7 Sensitivity 37%38% 36% 36% 38% 35% 36% 38% 35% Specificity 85% 93% 81% 84% 93% 80% 84%93% 81% OR Quartile 2 2.71 3.00 2.35 2.36 2.35 2.10 2.36 2.35 2.37 pValue 0.020 0.0074 0.058 0.040 0.034 0.091 0.040 0.034 0.049 Lower limitof 95% CI 1.17 1.34 0.970 1.04 1.07 0.888 1.04 1.07 1.00 Upper limit of95% CI 6.24 6.71 5.71 5.35 5.19 4.95 5.35 5.19 5.58 OR Quartile 3 2.203.57 2.18 2.07 3.16 2.15 2.07 3.16 2.26 p Value 0.025 5.8E−4 0.033 0.0390.0018 0.035 0.039 0.0018 0.023 Lower limit of 95% CI 1.10 1.73 1.061.04 1.53 1.06 1.04 1.53 1.12 Upper limit of 95% CI 4.40 7.36 4.45 4.126.51 4.37 4.12 6.51 4.56 OR Quartile 4 3.14 8.12 2.39 3.01 7.50 2.183.01 7.50 2.25 p Value 0.0064 2.3E−4 0.031 0.0086 3.9E−4 0.053 0.00863.9E−4 0.044 Lower limit of 95% CI 1.38 2.67 1.08 1.32 2.46 0.991 1.322.46 1.02 Upper limit of 95% CI 7.14 24.8 5.29 6.84 22.9 4.81 6.84 22.94.96

Example 6. Use of Chitinase-3-Like Protein 1 for Evaluating Renal Statusin Patients Admitted to the ICU: Recovery to RIFLE 0 and R from RIFLE Iand F

Patients from the intensive care unit (ICU) with RIFLE stage of injury(I) or failure (F) are enrolled in the following study. EDTAanti-coagulated blood samples (10 mL) and urine samples (50 mL) arecollected from each patient at enrollment, and at every 12 hours up today 3, and then every 24 hours thereafter up to day 7 while the subjectis hospitalized. Chitinase-3-like protein 1 is measured in theenrollment samples by standard immunoassay methods using commerciallyavailable assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine,urine output, or both serum creatinine and urine output during a periodstarting at 12, 24, 48, or 72 hours after sample collection or at anytime within 7 days after sample collection. Two cohorts are defined torepresent a “recovered” and a “non-recovered” population. “Recovered”indicates those patients whose maximum RIFLE stage during a period of24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R).“Non-recovered” indicates those patients whose maximum RIFLE stageduring a period of 24, 48 or 72 hours is injury (I) or failure (F). If apatient dies or is placed on renal replacement therapy (RRT) within 9days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohortsis determined using receiver operating characteristic (ROC) analysis.

TABLE 6 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts at 12 hours after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 13.4 32.3 11.9 32.6 10.4 32.9 Average 17.5 64.815.8 64.7 14.7 64.5 Stdev 19.6 80.5 18.0 80.1 17.8 79.8 p (t-test)0.0061 0.0052 0.0053 Min 0.372 0.0130 0.372 0.0130 0.372 0.0130 Max 64.6438 64.6 438 64.6 438 n (Patient) 23 111 22 112 21 113 sCr only Median12.8 33.5 12.8 33.5 12.2 34.1 Average 20.4 68.0 20.4 68.0 19.8 67.7Stdev 23.7 82.7 23.7 82.7 23.9 82.4 p (t-test) 0.0017 0.0017 0.0018 Min0.372 0.0130 0.372 0.0130 0.372 0.0130 Max 95.8 438 95.8 438 95.8 438 n(Patient) 32 102 32 102 31 103 UO only Median 15.0 41.3 15.0 46.1 14.741.3 Average 39.6 74.2 39.4 74.0 39.6 73.2 Stdev 55.2 89.4 55.6 88.756.0 88.3 p (t-test) 0.0078 0.0077 0.0096 Min 0.0130 0.584 0.0130 0.5840.0130 0.584 Max 237 438 237 438 237 438 n (Patient) 68 66 67 67 66 68Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr orUO sCr only UO only sCr or UO sCr only UO only AUC 0.66 0.66 0.63 0.680.66 0.64 0.69 0.67 0.64 SE 0.057 0.052 0.048 0.057 0.052 0.048 0.0570.052 0.048 p Value 0.0051 0.0019 0.0058 0.0020 0.0019 0.0039 9.1E−40.0011 0.0037 nCohort Recovered 23 32 68 22 32 67 21 31 66 nCohortNon-recovered 111 102 66 112 102 67 113 103 68 Cutoff Quartile 2 5.265.26 5.26 5.26 5.26 5.26 5.26 5.26 5.26 Sensitivity 74% 75% 80% 74% 75%81% 74% 76% 81% Specificity 22% 28% 31% 23% 28% 31% 24% 29% 32% CutoffQuartile 3 23.6 23.6 23.6 23.6 23.6 23.6 23.6 23.6 23.6 Sensitivity 56%57% 62% 56% 57% 63% 57% 57% 63% Specificity 78% 72% 62% 82% 72% 63% 86%74% 64% Cutoff Quartile 4 92.7 92.7 92.7 92.7 92.7 92.7 92.7 92.7 92.7Sensitivity 31% 32% 35% 30% 32% 34% 30% 32% 34% Specificity 100%  97%84% 100%  97% 84% 100%  97% 83% OR Quartile 2 0.785 1.21 1.82 0.842 1.211.90 0.905 1.28 1.97 p Value 0.66 0.68 0.14 0.76 0.68 0.11 0.86 0.590.094 Lower limit of 95% CI 0.267 0.493 0.822 0.285 0.493 0.856 0.3050.521 0.891 Upper limit of 95% CI 2.31 2.94 4.04 2.49 2.94 4.20 2.693.13 4.38 OR Quartile 3 4.56 3.37 2.65 5.79 3.37 2.82 7.84 3.86 3.01 pValue 0.0050 0.0059 0.0062 0.0027 0.0059 0.0037 0.0016 0.0031 0.0021Lower limit of 95% CI 1.58 1.42 1.32 1.84 1.42 1.40 2.18 1.58 1.49 Upperlimit of 95% CI 13.1 8.00 5.32 18.2 8.00 5.68 28.1 9.43 6.08 OR Quartile4 20.9 14.8 2.77 19.8 14.8 2.66 18.7 14.1 2.56 p Value 0.035 0.00940.015 0.039 0.0094 0.019 0.043 0.011 0.025 Lower limit of 95% CI 1.231.94 1.22 1.17 1.94 1.17 1.10 1.85 1.13 Upper limit of 95% CI 354 1136.30 335 113 6.04 317 108 5.80

TABLE 7 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts at 24 hours after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 11.3 35.9 8.66 37.1 8.66 37.1 Average 16.9 70.214.9 69.7 14.9 69.7 Stdev 19.4 82.8 18.2 82.1 18.2 82.1 p (t-test)3.0E−4 2.7E−4 2.7E−4 Min 0.0130 0.0690 0.0130 0.0690 0.0130 0.0690 Max64.6 438 64.6 438 64.6 438 n (Patient) 34 100 32 102 32 102 sCr onlyMedian 10.4 36.4 8.66 37.1 8.66 37.1 Average 18.7 72.2 18.2 71.9 18.271.9 Stdev 23.0 84.1 23.1 83.7 23.1 83.7 p (t-test) 1.5E−4 1.6E−4 1.6E−4Min 0.0130 0.0690 0.0130 0.0690 0.0130 0.0690 Max 95.8 438 95.8 438 95.8438 n (Patient) 39 95 38 96 38 96 UO only Median 15.1 46.1 15.0 46.115.0 46.1 Average 40.5 74.9 40.5 73.9 40.5 73.9 Stdev 54.8 89.6 55.588.4 55.5 88.4 p (t-test) 0.0088 0.011 0.011 Min 0.0130 0.584 0.01300.584 0.0130 0.584 Max 234 438 234 438 234 438 n (Patient) 68 61 66 6366 63 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO onlysCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.71 0.630.73 0.72 0.63 0.73 0.72 0.63 SE 0.047 0.046 0.049 0.046 0.046 0.0490.046 0.046 0.049 p Value 9.8E−6 4.7E−6 0.0092 7.5E−7 2.0E−6 0.00647.5E−7 2.0E−6 0.0064 nCohort Recovered 34 39 68 32 38 66 32 38 66nCohort Non-recovered 100 95 61 102 96 63 102 96 63 Cutoff Quartile 25.26 5.26 5.34 5.26 5.26 5.34 5.26 5.26 5.34 Sensitivity 77% 79% 80% 77%79% 81% 77% 79% 81% Specificity 32% 36% 29% 34% 37% 30% 34% 37% 30%Cutoff Quartile 3 23.6 23.6 25.7 23.6 23.6 25.7 23.6 23.6 25.7Sensitivity 59% 60% 61% 60% 60% 62% 60% 60% 62% Specificity 76% 74% 59%81% 76% 61% 81% 76% 61% Cutoff Quartile 4 92.7 92.7 92.9 92.7 92.7 92.992.7 92.7 92.9 Sensitivity 34% 35% 36% 33% 34% 35% 33% 34% 35%Specificity 100%  97% 84% 100%  97% 83% 100%  97% 83% OR Quartile 2 1.602.10 1.70 1.80 2.22 1.85 1.80 2.22 1.85 p Value 0.28 0.076 0.20 0.180.058 0.14 0.18 0.058 0.14 Lower limit of 95% CI 0.680 0.925 0.750 0.7580.974 0.815 0.758 0.974 0.815 Upper limit of 95% CI 3.77 4.77 3.86 4.275.05 4.19 4.27 5.05 4.19 OR Quartile 3 4.68 4.35 2.20 6.45 4.92 2.506.45 4.92 2.50 p Value 6.5E−4 5.0E−4 0.028 1.7E−4 2.5E−4 0.011 1.7E−42.5E−4 0.011 Lower limit of 95% CI 1.93 1.90 1.09 2.44 2.10 1.23 2.442.10 1.23 Upper limit of 95% CI 11.4 9.95 4.46 17.0 11.5 5.08 17.0 11.55.08 OR Quartile 4 35.8 20.2 2.92 32.7 19.4 2.68 32.7 19.4 2.68 p Value0.013 0.0037 0.011 0.015 0.0042 0.020 0.015 0.0042 0.020 Lower limit of95% CI 2.13 2.66 1.27 1.95 2.54 1.17 1.95 2.54 1.17 Upper limit of 95%CI 602 154 6.71 551 148 6.15 551 148 6.15

TABLE 8 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts at 48 hours after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 10.8 37.8 10.8 37.8 10.8 37.8 Average 24.7 74.024.7 74.0 24.7 74.0 Stdev 32.7 86.3 32.7 86.3 32.7 86.3 p (t-test)2.8E−4 2.8E−4 2.8E−4 Min 0.0130 0.0690 0.0130 0.0690 0.0130 0.0690 Max136 438 136 438 136 438 n (Patient) 46 87 46 87 46 87 sCr only Median11.7 37.1 11.7 37.1 11.7 37.1 Average 26.9 76.0 26.9 76.0 26.9 76.0Stdev 33.4 88.1 33.4 88.1 33.4 88.1 p (t-test) 2.5E−4 2.5E−4 2.5E−4 Min0.0130 0.0690 0.0130 0.0690 0.0130 0.0690 Max 136 438 136 438 136 438 n(Patient) 50 82 50 82 50 82 UO only Median 15.2 46.1 15.2 46.1 15.2 41.3Average 46.1 73.3 46.1 73.3 46.3 72.6 Stdev 61.4 89.1 61.4 89.1 61.988.4 p (t-test) 0.052 0.052 0.060 Min 0.0130 0.584 0.0130 0.584 0.01300.584 Max 237 438 237 438 237 438 n (Patient) 63 57 63 57 62 58 RecoveryPeriod Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCronly UO only sCr or UO sCr only UO only AUC 0.69 0.68 0.61 0.69 0.680.61 0.69 0.68 0.61 SE 0.046 0.047 0.052 0.046 0.047 0.052 0.046 0.0470.051 p Value 3.6E−5 1.4E−4 0.027 3.6E−5 1.4E−4 0.027 3.6E−5 1.4E−40.025 nCohort Recovered 46 50 63 46 50 63 46 50 62 nCohort Non-recovered87 82 57 87 82 57 87 82 58 Cutoff Quartile 2 5.24 5.31 5.63 5.24 5.315.63 5.24 5.31 5.63 Sensitivity 80% 80% 81% 80% 80% 81% 80% 80% 81%Specificity 35% 34% 30% 35% 34% 30% 35% 34% 31% Cutoff Quartile 3 25.125.4 28.5 25.1 25.4 28.5 25.1 25.4 28.5 Sensitivity 61% 60% 60% 61% 60%60% 61% 60% 60% Specificity 70% 66% 59% 70% 66% 59% 70% 66% 60% CutoffQuartile 4 92.9 93.6 96.0 92.9 93.6 96.0 92.9 93.6 96.0 Sensitivity 37%37% 33% 37% 37% 33% 37% 37% 33% Specificity 96% 94% 83% 96% 94% 83% 96%94% 82% OR Quartile 2 2.20 2.12 1.81 2.20 2.12 1.81 2.20 2.12 1.89 pValue 0.056 0.065 0.17 0.056 0.065 0.17 0.056 0.065 0.14 Lower limit of95% CI 0.981 0.954 0.772 0.981 0.954 0.772 0.981 0.954 0.807 Upper limitof 95% CI 4.91 4.73 4.22 4.91 4.73 4.22 4.91 4.73 4.42 OR Quartile 33.56 2.88 2.10 3.56 2.88 2.10 3.56 2.88 2.25 p Value 0.0011 0.0046 0.0460.0011 0.0046 0.046 0.0011 0.0046 0.029 Lower limit of 95% CI 1.66 1.381.01 1.66 1.38 1.01 1.66 1.38 1.08 Upper limit of 95% CI 7.63 6.00 4.367.63 6.00 4.36 7.63 6.00 4.68 OR Quartile 4 12.8 9.04 2.36 12.8 9.042.36 12.8 9.04 2.26 p Value 7.5E−4 5.6E−4 0.048 7.5E−4 5.6E−4 0.0487.5E−4 5.6E−4 0.061 Lower limit of 95% CI 2.91 2.59 1.01 2.91 2.59 1.012.91 2.59 0.964 Upper limit of 95% CI 56.4 31.6 5.54 56.4 31.6 5.54 56.431.6 5.29

TABLE 9 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts at 72 hours after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 10.8 47.5 10.8 47.2 11.3 47.0 Average 25.0 81.925.2 80.2 25.6 79.2 Stdev 32.2 90.0 32.6 89.4 32.8 89.3 p (t-test)1.4E−5 3.1E−5 5.4E−5 Min 0.0130 0.129 0.0130 0.129 0.0130 0.129 Max 136438 136 438 136 438 n (Patient) 56 74 54 76 53 77 sCr only Median 11.747.2 11.7 47.2 11.7 47.2 Average 26.8 82.0 26.8 82.0 26.8 82.0 Stdev33.2 91.2 33.2 91.2 33.2 91.2 p (t-test) 2.4E−5 2.4E−5 2.4E−5 Min 0.01300.129 0.0130 0.129 0.0130 0.129 Max 136 438 136 438 136 438 n (Patient)58 72 58 72 58 72 UO only Median 18.1 47.2 18.1 41.3 19.9 36.4 Average47.3 78.9 49.3 74.4 50.2 73.1 Stdev 61.7 92.8 63.2 90.8 63.5 90.4 p(t-test) 0.033 0.092 0.12 Min 0.0130 0.584 0.0130 0.584 0.0130 0.584 Max237 438 237 438 237 438 n (Patient) 62 50 58 54 57 55 Recovery PeriodDuration (hr) 24 48 72 sCr or sCr or sCr or UO sCr only UO only UO sCronly UO only UO sCr only UO only AUC 0.72 0.70 0.62 0.71 0.70 0.60 0.700.70 0.59 SE 0.044 0.045 0.054 0.045 0.045 0.054 0.045 0.045 0.054 pValue 8.7E−7 7.6E−6 0.027 1.7E−6 7.6E−6 0.069 5.9E−6 7.6E−6 0.11 nCohortRecovered 56 58 62 54 58 58 53 58 57 nCohort Non-recovered 74 72 50 7672 54 77 72 55 Cutoff Quartile 2 5.36 5.36 5.63 5.36 5.36 5.63 5.36 5.365.63 Sensitivity 84% 83% 84% 83% 83% 81% 82% 83% 80% Specificity 38% 36%32% 37% 36% 31% 36% 36% 30% Cutoff Quartile 3 25.4 25.4 30.7 25.4 25.430.7 25.4 25.4 30.7 Sensitivity 64% 62% 62% 63% 62% 59% 62% 62% 58%Specificity 68% 66% 60% 69% 66% 59% 68% 66% 58% Cutoff Quartile 4 92.792.7 97.8 92.7 92.7 97.8 92.7 92.7 97.8 Sensitivity 41% 40% 34% 39% 40%31% 39% 40% 31% Specificity 95% 93% 82% 94% 93% 81% 94% 93% 81% ORQuartile 2 3.10 2.84 2.50 2.85 2.84 1.98 2.51 2.84 1.70 p Value 0.00690.013 0.052 0.012 0.013 0.13 0.025 0.013 0.23 Lower limit of 95% CI 1.361.25 0.992 1.26 1.25 0.818 1.12 1.25 0.712 Upper limit of 95% CI 7.056.44 6.30 6.43 6.44 4.79 5.63 6.44 4.06 OR Quartile 3 3.67 3.17 2.413.73 3.17 2.06 3.51 3.17 1.91 p Value 5.1E−4 0.0017 0.024 4.8E−4 0.00170.060 8.7E−4 0.0017 0.090 Lower limit of 95% CI 1.76 1.54 1.12 1.78 1.540.970 1.68 1.54 0.903 Upper limit of 95% CI 7.66 6.52 5.18 7.82 6.524.38 7.33 6.52 4.05 OR Quartile 4 12.0 9.10 2.39 11.1 9.10 1.96 10.69.10 1.87 p Value 9.8E−5 1.1E−4 0.051 1.7E−4 1.1E−4 0.13 2.1E−4 1.1E−40.16 Lower limit of 95% CI 3.44 2.97 0.995 3.17 2.97 0.821 3.04 2.970.782 Upper limit of 95% CI 42.1 27.9 5.73 38.8 27.9 4.70 37.2 27.9 4.47

TABLE 10 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “recovered” and “non-recovered” cohortswhere recovery starts within 7 days after sample collection and renalstatus is assessed by serum creatinine (sCr) only, urine output (UO)only, or serum creatinine or urine output RIFLE criteria. RecoveryPeriod Duration (hr) 24 48 72 Recovered Non-recovered RecoveredNon-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort CohortCohort sCr or UO Median 15.1 47.2 15.1 47.0 15.1 46.5 Average 38.7 81.738.7 80.9 39.2 79.6 Stdev 52.3 94.6 52.7 94.0 52.9 93.7 p (t-test)1.0E−3 0.0012 0.0020 Min 0.0130 0.584 0.0130 0.584 0.0130 0.584 Max 234438 234 438 234 438 n (Patient) 78 56 77 57 76 58 sCr only Median 13.647.5 13.6 47.5 13.6 47.5 Average 31.2 82.1 31.2 82.1 31.2 82.1 Stdev43.6 91.4 43.6 91.4 43.6 91.4 p (t-test) 6.8E−5 6.8E−5 6.8E−5 Min 0.01300.307 0.0130 0.307 0.0130 0.307 Max 234 438 234 438 234 438 n (Patient)67 67 67 67 67 67 UO only Median 15.2 46.1 15.2 46.1 15.2 36.4 Average44.5 79.2 44.5 79.2 45.1 76.7 Stdev 59.5 95.9 59.5 95.9 60.0 94.7 p(t-test) 0.011 0.011 0.019 Min 0.0130 0.584 0.0130 0.584 0.0130 0.584Max 237 438 237 438 237 438 n (Patient) 87 47 87 47 85 49 RecoveryPeriod Duration (hr) 24 48 72 sCr or sCr or sCr or UO sCr only UO onlyUO sCr only UO only UO sCr only UO only AUC 0.65 0.68 0.62 0.65 0.680.62 0.64 0.68 0.61 SE 0.049 0.046 0.052 0.049 0.046 0.052 0.049 0.0460.051 p Value 0.0027 5.9E−5 0.025 0.0023 5.9E−5 0.025 0.0042 5.9E−50.037 nCohort Recovered 78 67 87 77 67 87 76 67 85 nCohort Non-recovered56 67 47 57 67 47 58 67 49 Cutoff Quartile 2 5.26 5.26 5.26 5.26 5.265.26 5.26 5.26 5.26 Sensitivity 84% 84% 83% 84% 84% 83% 83% 84% 82%Specificity 32% 34% 30% 32% 34% 30% 32% 34% 29% Cutoff Quartile 3 23.623.6 23.6 23.6 23.6 23.6 23.6 23.6 23.6 Sensitivity 61% 63% 60% 61% 63%60% 60% 63% 59% Specificity 58% 63% 55% 58% 63% 55% 58% 63% 55% CutoffQuartile 4 92.7 92.7 92.7 92.7 92.7 92.7 92.7 92.7 92.7 Sensitivity 39%42% 38% 39% 42% 38% 38% 42% 37% Specificity 85% 91% 82% 84% 91% 82% 84%91% 81% OR Quartile 2 2.46 2.66 2.08 2.56 2.66 2.08 2.22 2.66 1.85 pValue 0.039 0.019 0.11 0.031 0.019 0.11 0.062 0.019 0.16 Lower limit of95% CI 1.05 1.17 0.855 1.09 1.17 0.855 0.961 1.17 0.783 Upper limit of95% CI 5.80 6.04 5.05 6.04 6.04 5.05 5.11 6.04 4.38 OR Quartile 3 2.112.82 1.81 2.24 2.82 1.81 2.09 2.82 1.79 p Value 0.037 0.0037 0.10 0.0240.0037 0.10 0.038 0.0037 0.11 Lower limit of 95% CI 1.05 1.40 0.883 1.111.40 0.883 1.04 1.40 0.880 Upper limit of 95% CI 4.24 5.68 3.73 4.505.68 3.73 4.20 5.68 3.66 OR Quartile 4 3.56 7.30 2.75 3.40 7.30 2.753.26 7.30 2.50 p Value 0.0023 5.8E−5 0.013 0.0032 5.8E−5 0.013 0.00445.8E−5 0.024 Lower limit of 95% CI 1.57 2.77 1.24 1.51 2.77 1.24 1.452.77 1.13 Upper limit of 95% CI 8.05 19.2 6.13 7.69 19.2 6.13 7.35 19.25.55

Example 7. Use of Chitinase-3-Like Protein 1 for Evaluating Renal Statusin Patients Admitted to the ICU: Persistent at RIFLE F

Patients from the intensive care unit (ICU) with RIFLE stage of injury(I) or failure (F) are enrolled in the following study. EDTAanti-coagulated blood samples (10 mL) and urine samples (50 mL) arecollected from each patient at enrollment, and at every 12 hours up today 3, and then every 24 hours thereafter up to day 7 while the subjectis hospitalized. Chitinase-3-like protein 1 is measured in theenrollment samples by standard immunoassay methods using commerciallyavailable assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine,urine output, or both serum creatinine and urine output. Two cohorts aredefined to represent a “persistent” and a “non-persistent” population.“Persistent” indicates those patients whose minimum RIFLE stage during aperiod of 24, 48 or 72 hours is failure (F) where the persistence periodcan start from the time of sample collection to 24, 48, 72, 96 or 168hours after sample collection. “Non-persistent” indicates those patientswho are not persistent at failure (F) and whose minimum RIFLE stageduring a period of 24, 48 or 72 hours is non-injury (RIFLE 0), risk ofinjury (R), or injury (I) where the persistence period can start fromthe time of sample collection to 24, 48, 72, 96 or 168 hours aftersample collection. If a patient dies after failure (F) or is placed onrenal replacement therapy (RRT) at any time from sample collection to24, 48, 72, 96 or 168 hours after sample collection, the patient isconsidered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohortsis determined using receiver operating characteristic (ROC) analysis.

TABLE 11 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 24 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 13.2 73.9 11.3 97.8 13.5 92.1Average 36.1 90.2 36.3 99.7 45.7 82.0 Stdev 54.3 95.4 56.3 95.7 74.670.3 p (t-test) 2.0E−5 1.6E−6 0.012 Min 0.0130 0.819 0.0130 0.819 0.01300.819 Max 298 438 298 438 438 237 n (Patient) 99 50 107 42 114 35 sCronly Median 13.2 55.6 11.7 92.5 13.4 50.6 Average 35.6 88.4 40.3 88.046.2 76.6 Stdev 54.2 95.3 68.6 80.6 75.5 70.2 p (t-test) 3.2E−5 3.9E−40.033 Min 0.0130 0.584 0.0130 0.584 0.0130 0.584 Max 298 438 438 338 438237 n (Patient) 97 51 106 42 111 37 UO only Median 15.2 92.1 15.6 10115.6 101 Average 50.6 85.1 50.6 87.5 50.6 87.5 Stdev 74.8 72.9 74.5 74.674.5 74.6 p (t-test) 0.074 0.063 0.063 Min 0.0130 0.819 0.0130 0.8190.0130 0.819 Max 438 231 438 231 438 231 n (Patient) 131 17 132 16 13216 Persistence Period Duration (hr) 24 48 72 sCr or sCr or sCr or UO sCronly UO only UO sCr only UO only UO sCr only UO only AUC 0.70 0.69 0.660.75 0.72 0.66 0.70 0.67 0.66 SE 0.047 0.047 0.076 0.048 0.050 0.0780.054 0.054 0.078 p Value 1.8E−5 5.9E−5 0.033 1.5E−7 1.1E−5 0.040 3.0E−40.0016 0.040 nCohort Non-persistent 99 97 131 107 106 132 114 111 132nCohort Persistent 50 51 17 42 42 16 35 37 16 Cutoff Quartile 2 3.433.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56 Sensitivity 86% 84% 88% 93% 90%88% 91% 89% 88% Specificity 30% 30% 27% 32% 31% 27% 30% 30% 27% CutoffQuartile 3 17.6 16.9 17.8 17.6 16.9 17.8 17.6 16.9 17.8 Sensitivity 68%67% 71% 76% 74% 69% 71% 70% 69% Specificity 59% 59% 53% 60% 59% 52% 56%57% 52% Cutoff Quartile 4 89.0 88.4 89.4 89.0 88.4 89.4 89.0 88.4 89.4Sensitivity 50% 49% 53% 57% 52% 56% 51% 46% 56% Specificity 87% 88% 79%87% 86% 79% 82% 82% 79% OR Quartile 2 2.67 2.29 2.73 6.05 4.29 2.53 4.533.49 2.53 p Value 0.034 0.062 0.20 0.0045 0.010 0.24 0.018 0.028 0.24Lower limit of 95% CI 1.08 0.959 0.595 1.75 1.42 0.546 1.30 1.14 0.546Upper limit of 95% CI 6.61 5.48 12.6 21.0 13.0 11.7 15.8 10.6 11.7 ORQuartile 3 3.01 2.85 2.67 4.76 4.13 2.41 3.20 3.10 2.41 p Value 0.00260.0038 0.080 1.5E−4 4.3E−4 0.12 0.0055 0.0055 0.12 Lower limit of 95% CI1.47 1.40 0.891 2.12 1.87 0.793 1.41 1.40 0.793 Upper limit of 95% CI6.15 5.79 8.01 10.7 9.09 7.32 7.28 6.90 7.32 OR Quartile 4 6.62 6.814.14 8.86 6.67 4.78 4.98 3.87 4.78 p Value 4.2E−6 4.1E−6 0.0074 2.6E−75.1E−6 0.0043 1.3E−4 0.0010 0.0043 Lower limit of 95% CI 2.96 3.01 1.463.86 2.95 1.63 2.19 1.72 1.63 Upper limit of 95% CI 14.8 15.4 11.7 20.315.1 14.0 11.3 8.67 14.0

TABLE 12 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 48 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 11.3 73.9 10.1 96.7 12.2 73.9Average 32.3 88.6 32.7 95.6 40.4 87.1 Stdev 52.0 91.5 54.7 90.5 65.685.9 p (t-test) 4.1E−6 4.4E−7 4.3E−4 Min 0.0130 0.307 0.0130 0.8190.0130 0.819 Max 298 438 298 438 338 438 n (Patient) 91 58 98 51 105 44sCr only Median 12.2 50.6 10.4 92.1 13.2 47.5 Average 33.4 86.5 33.991.7 41.9 81.1 Stdev 52.3 93.0 55.2 92.2 66.3 87.1 p (t-test) 1.7E−54.5E−6 0.0032 Min 0.0130 0.307 0.0130 0.584 0.0130 0.584 Max 298 438 298438 338 438 n (Patient) 91 57 97 51 103 45 UO only Median 15.0 92.1 15.094.8 15.0 94.8 Average 46.6 93.6 46.7 95.5 46.7 95.5 Stdev 67.2 98.666.9 100 66.9 100 p (t-test) 0.0041 0.0033 0.0033 Min 0.0130 0.8190.0130 0.819 0.0130 0.819 Max 338 438 338 438 338 438 n (Patient) 123 25124 24 124 24 Persistence Period Duration (hr) 24 48 72 sCr or sCr orsCr or UO sCr only UO only UO sCr only UO only UO sCr only UO only AUC0.72 0.70 0.68 0.77 0.74 0.68 0.72 0.68 0.68 SE 0.044 0.046 0.063 0.0430.045 0.064 0.049 0.050 0.064 p Value 6.2E−7 1.8E−5 0.0049 5.7E−101.5E−7 0.0060 7.1E−6 2.7E−4 0.0060 nCohort Non-persistent 91 91 123 9897 124 105 103 124 nCohort Persistent 58 57 25 51 51 24 44 45 24 CutoffQuartile 2 3.43 3.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56 Sensitivity 86%84% 88% 92% 90% 88% 91% 89% 88% Specificity 32% 31% 28% 34% 33% 27% 31%31% 27% Cutoff Quartile 3 17.6 16.9 17.8 17.6 16.9 17.8 17.6 16.9 17.8Sensitivity 69% 67% 72% 76% 73% 71% 73% 69% 71% Specificity 62% 60% 54%63% 62% 54% 59% 58% 54% Cutoff Quartile 4 89.0 88.4 89.4 89.0 88.4 89.489.0 88.4 89.4 Sensitivity 50% 47% 52% 55% 51% 54% 50% 44% 54%Specificity 90% 89% 80% 90% 89% 81% 85% 83% 81% OR Quartile 2 2.92 2.372.80 5.97 4.53 2.64 4.58 3.61 2.64 p Value 0.015 0.044 0.11 0.00150.0035 0.13 0.0070 0.014 0.13 Lower limit of 95% CI 1.23 1.02 0.787 1.981.64 0.741 1.51 1.30 0.741 Upper limit of 95% CI 6.96 5.49 9.97 18.012.5 9.44 13.9 9.99 9.44 OR Quartile 3 3.56 3.06 3.08 5.60 4.29 2.853.84 3.09 2.85 p Value 3.7E−4 0.0016 0.019 1.1E−5 1.1E−4 0.030 6.0E−40.0029 0.030 Lower limit of 95% CI 1.77 1.53 1.20 2.60 2.05 1.11 1.781.47 1.11 Upper limit of 95% CI 7.15 6.11 7.89 12.0 8.97 7.37 8.30 6.497.37 OR Quartile 4 9.11 7.29 4.47 10.7 8.13 4.92 5.56 4.05 4.92 p Value4.7E−7 3.4E−6 0.0011 5.6E−8 8.4E−7 6.7E−4 2.3E−5 4.8E−4 6.7E−4 Lowerlimit of 95% CI 3.86 3.15 1.81 4.55 3.53 1.97 2.51 1.85 1.97 Upper limitof 95% CI 21.5 16.8 11.0 25.2 18.7 12.3 12.3 8.87 12.3

TABLE 13 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 72 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 10.8 50.6 10.1 92.1 12.7 47.5Average 30.8 86.1 29.6 91.8 39.2 83.1 Stdev 48.6 91.6 47.9 92.0 60.391.1 p (t-test) 4.7E−6 2.6E−7 5.6E−4 Min 0.0130 0.307 0.0130 0.8190.0130 0.819 Max 298 438 298 438 298 438 n (Patient) 86 63 90 59 98 51sCr only Median 11.3 47.5 10.4 73.9 13.2 47.0 Average 31.2 84.3 29.990.9 39.7 80.7 Stdev 48.8 92.2 47.8 93.2 60.5 91.9 p (t-test) 1.2E−55.6E−7 0.0014 Min 0.0130 0.307 0.0130 0.584 0.0130 0.584 Max 298 438 298438 298 438 n (Patient) 85 63 90 58 97 51 UO only Median 14.7 46.8 15.046.1 15.0 46.1 Average 45.2 88.7 45.2 90.0 45.2 90.0 Stdev 63.0 103 62.7104 62.7 104 p (t-test) 0.0034 0.0029 0.0029 Min 0.0130 0.819 0.01300.819 0.0130 0.819 Max 298 438 298 438 298 438 n (Patient) 116 32 117 31117 31 Persistence Period Duration (hr) 24 48 72 sCr or sCr or sCr or UOsCr only UO only UO sCr only UO only UO sCr only UO only AUC 0.71 0.690.65 0.75 0.74 0.65 0.69 0.67 0.65 SE 0.044 0.044 0.058 0.042 0.0430.059 0.047 0.048 0.059 p Value 1.2E−6 1.2E−5 0.010 1.5E−9 5.3E−8 0.0135.8E−5 4.6E−4 0.013 nCohort Non-persistent 86 85 116 90 90 117 98 97 117nCohort Persistent 63 63 32 59 58 31 51 51 31 Cutoff Quartile 2 3.433.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56 Sensitivity 86% 84% 84% 92% 90%84% 90% 88% 84% Specificity 33% 32% 28% 36% 34% 27% 33% 32% 27% CutoffQuartile 3 17.6 16.9 17.8 17.6 16.9 17.8 17.6 16.9 17.8 Sensitivity 68%67% 69% 73% 71% 68% 69% 67% 68% Specificity 63% 62% 55% 64% 63% 55% 59%59% 55% Cutoff Quartile 4 89.0 88.4 89.4 89.0 88.4 89.4 89.0 88.4 89.4Sensitivity 48% 46% 47% 51% 50% 48% 45% 43% 48% Specificity 91% 91% 81%91% 91% 81% 85% 85% 81% OR Quartile 2 2.90 2.47 2.06 5.96 4.55 1.96 4.463.52 1.96 p Value 0.013 0.030 0.17 5.5E−4 0.0018 0.21 0.0039 0.0096 0.21Lower limit of 95% CI 1.25 1.09 0.729 2.16 1.76 0.692 1.62 1.36 0.692Upper limit of 95% CI 6.69 5.58 5.81 16.4 11.8 5.54 12.3 9.13 5.54 ORQuartile 3 3.63 3.31 2.71 4.87 4.17 2.54 3.17 2.85 2.54 p Value 2.4E−45.9E−4 0.019 1.6E−5 8.1E−5 0.029 0.0016 0.0038 0.029 Lower limit of 95%CI 1.82 1.67 1.18 2.38 2.05 1.10 1.55 1.40 1.10 Upper limit of 95% CI7.22 6.56 6.22 9.99 8.47 5.85 6.49 5.79 5.85 OR Quartile 4 8.86 8.213.77 10.6 10.2 4.05 4.55 4.15 4.05 p Value 1.2E−6 2.8E−6 0.0018 1.8E−73.0E−7 0.0012 1.4E−4 3.6E−4 0.0012 Lower limit of 95% CI 3.68 3.40 1.644.37 4.21 1.74 2.09 1.90 1.74 Upper limit of 95% CI 21.4 19.8 8.69 25.825.0 9.41 9.90 9.06 9.41

TABLE 14 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 96 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 10.4 53.1 10.1 92.1 11.7 47.5Average 30.1 86.3 29.6 91.8 38.8 82.1 Stdev 48.4 90.9 47.9 92.0 60.789.8 p (t-test) 3.0E−6 2.6E−7 6.2E−4 Min 0.0130 0.307 0.0130 0.8190.0130 0.819 Max 298 438 298 438 298 438 n (Patient) 85 64 90 59 96 53sCr only Median 11.3 47.5 10.4 55.6 12.2 47.0 Average 31.2 84.3 29.989.9 39.2 80.0 Stdev 48.8 92.2 48.1 92.7 60.9 90.4 p (t-test) 1.2E−58.0E−7 0.0013 Min 0.0130 0.307 0.0130 0.584 0.0130 0.584 Max 298 438 298438 298 438 n (Patient) 85 63 89 59 95 53 UO only Median 14.1 46.5 14.741.3 14.7 41.3 Average 45.1 84.0 45.6 84.8 45.6 84.8 Stdev 63.8 98.163.4 101 63.4 101 p (t-test) 0.0064 0.0072 0.0072 Min 0.0130 0.8190.0130 0.819 0.0130 0.819 Max 298 438 298 438 298 438 n (Patient) 112 36114 34 114 34 Persistence Period Duration (hr) 24 48 72 sCr or sCr orsCr or UO sCr only UO only UO sCr only UO only UO sCr only UO only AUC0.72 0.69 0.65 0.75 0.74 0.64 0.69 0.68 0.64 SE 0.043 0.044 0.055 0.0420.043 0.057 0.047 0.047 0.057 p Value 4.2E−7 1.2E−5 0.0065 1.5E−9 3.9E−80.013 2.9E−5 2.0E−4 0.013 nCohort Non-persistent 85 85 112 90 89 114 9695 114 nCohort Persistent 64 63 36 59 59 34 53 53 34 Cutoff Quartile 23.43 3.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56 Sensitivity 86% 84% 86% 92%90% 85% 91% 89% 85% Specificity 33% 32% 29% 36% 35% 28% 33% 33% 28%Cutoff Quartile 3 17.6 16.9 17.8 17.6 16.9 17.8 17.6 16.9 17.8Sensitivity 69% 67% 69% 73% 71% 68% 70% 68% 68% Specificity 64% 62% 56%64% 64% 55% 60% 60% 55% Cutoff Quartile 4 89.0 88.4 89.4 89.0 88.4 89.489.0 88.4 89.4 Sensitivity 48% 46% 44% 51% 49% 44% 45% 43% 44%Specificity 92% 91% 81% 91% 91% 81% 85% 85% 81% OR Quartile 2 3.00 2.472.48 5.96 4.72 2.26 4.80 3.79 2.26 p Value 0.010 0.030 0.084 5.5E−40.0014 0.12 0.0024 0.0060 0.12 Lower limit of 95% CI 1.30 1.09 0.8862.16 1.83 0.805 1.74 1.46 0.805 Upper limit of 95% CI 6.94 5.58 6.9416.4 12.2 6.36 13.2 9.83 6.36 OR Quartile 3 3.83 3.31 2.92 4.87 4.402.58 3.53 3.18 2.58 p Value 1.3E−4 5.9E−4 0.0087 1.6E−5 4.4E−5 0.0215.5E−4 0.0014 0.021 Lower limit of 95% CI 1.92 1.67 1.31 2.38 2.16 1.151.73 1.56 1.15 Upper limit of 95% CI 7.63 6.56 6.51 9.99 8.96 5.79 7.226.45 5.79 OR Quartile 4 10.5 8.21 3.47 10.6 9.79 3.30 4.85 4.44 3.30 pValue 5.0E−7 2.8E−6 0.0027 1.8E−7 4.8E−7 0.0044 7.9E−5 2.0E−4 0.0044Lower limit of 95% CI 4.19 3.40 1.54 4.37 4.03 1.45 2.21 2.02 1.45 Upperlimit of 95% CI 26.2 19.8 7.80 25.8 23.8 7.51 10.6 9.73 7.51

TABLE 15 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 168 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 10.4 48.2 9.56 55.6 11.3 47.8Average 30.3 83.5 29.0 88.7 38.5 80.3 Stdev 49.1 89.8 48.3 90.2 61.288.1 p (t-test) 9.4E−6 6.4E−7 8.5E−4 Min 0.0130 0.307 0.0130 0.5840.0130 0.584 Max 298 438 298 438 298 438 n (Patient) 82 67 86 63 93 56sCr only Median 10.4 47.5 9.80 50.6 11.3 47.0 Average 31.1 82.9 29.788.2 39.3 78.4 Stdev 49.4 91.2 48.6 91.6 61.5 89.1 p (t-test) 1.9E−51.4E−6 0.0019 Min 0.0130 0.307 0.0130 0.584 0.0130 0.584 Max 298 438 298438 298 438 n (Patient) 83 65 87 61 93 55 UO only Median 13.6 47.0 14.146.5 14.1 46.5 Average 44.2 85.7 44.2 86.8 44.2 86.8 Stdev 63.4 97.263.1 98.4 63.1 98.4 p (t-test) 0.0033 0.0028 0.0028 Min 0.0130 0.8190.0130 0.819 0.0130 0.819 Max 298 438 298 438 298 438 n (Patient) 111 37112 36 112 36 Persistence Period Duration (hr) 24 48 72 sCr or sCr orsCr or UO sCr only UO only UO sCr only UO only UO sCr only UO only AUC0.71 0.70 0.66 0.75 0.74 0.66 0.69 0.68 0.66 SE 0.043 0.044 0.054 0.0420.043 0.055 0.046 0.047 0.055 p Value 1.2E−6 6.0E−6 0.0029 1.7E−9 1.6E−80.0037 3.7E−5 1.2E−4 0.0037 nCohort Non-persistent 82 83 111 86 87 11293 93 112 nCohort Persistent 67 65 37 63 61 36 56 55 36 Cutoff Quartile2 3.43 3.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56 Sensitivity 85% 85% 86%90% 90% 86% 89% 89% 86% Specificity 33% 33% 29% 36% 36% 29% 33% 33% 29%Cutoff Quartile 3 17.6 16.9 17.8 17.6 16.9 17.8 17.6 16.9 17.8Sensitivity 69% 68% 70% 73% 72% 69% 70% 69% 69% Specificity 65% 64% 57%66% 66% 56% 61% 61% 56% Cutoff Quartile 4 89.0 88.4 89.4 89.0 88.4 89.489.0 88.4 89.4 Sensitivity 46% 45% 46% 49% 48% 47% 45% 42% 47%Specificity 91% 90% 82% 92% 91% 82% 86% 85% 82% OR Quartile 2 2.80 2.652.59 5.35 5.07 2.48 4.17 4.08 2.48 p Value 0.013 0.019 0.069 5.3E−48.1E−4 0.084 0.0032 0.0037 0.084 Lower limit of 95% CI 1.24 1.17 0.9272.07 1.96 0.886 1.61 1.58 0.886 Upper limit of 95% CI 6.32 5.99 7.2513.8 13.1 6.94 10.8 10.6 6.94 OR Quartile 3 4.00 3.70 3.10 5.32 4.922.92 3.63 3.54 2.92 p Value 7.5E−5 1.9E−4 0.0055 4.4E−6 1.2E−5 0.00873.4E−4 4.7E−4 0.0087 Lower limit of 95% CI 2.01 1.86 1.40 2.61 2.41 1.311.79 1.74 1.31 Upper limit of 95% CI 7.95 7.35 6.90 10.9 10.0 6.51 7.367.18 6.51 OR Quartile 4 9.23 7.55 3.87 10.9 8.95 4.12 4.96 4.06 4.12 pValue 1.8E−6 6.4E−6 0.0010 3.2E−7 1.2E−6 6.5E−4 6.8E−5 4.4E−4 6.5E−4Lower limit of 95% CI 3.71 3.14 1.72 4.37 3.70 1.82 2.26 1.86 1.82 Upperlimit of 95% CI 23.0 18.2 8.67 27.4 21.7 9.29 10.9 8.86 9.29

Example 8. Use of Chitinase-3-Like Protein 1 for Evaluating Renal Statusin Patients Admitted to the ICU: Persistent at RIFLE I or F

Patients from the intensive care unit (ICU) with RIFLE stage of injury(I) or failure (F) are enrolled in the following study. EDTAanti-coagulated blood samples (10 mL) and urine samples (50 mL) arecollected from each patient at enrollment, and at every 12 hours up today 3, and then every 24 hours thereafter up to day 7 while the subjectis hospitalized. Chitinase-3-like protein 1 is measured in theenrollment samples by standard immunoassay methods using commerciallyavailable assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine,urine output, or both serum creatinine and urine output. Two cohorts aredefined to represent a “persistent” and a “non-persistent” population.“Persistent” indicates those patients whose minimum RIFLE stage during aperiod of 24, 48 or 72 hours is injury (I) or failure (F) where thepersistence period can start from the time of sample collection to 24,48, 72, 96 or 168 hours after sample collection. “Non-persistent”indicates those patients who are not persistent at injury (I) or failure(F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hoursis non-injury (RIFLE 0) or risk of injury (R) where the persistenceperiod can start from the time of sample collection to 24, 48, 72, 96 or168 hours after sample collection. If a patient dies after injury (I) orfailure (F) or is placed on renal replacement therapy (RRT) at any timefrom sample collection to 24, 48, 72, 96 or 168 hours after samplecollection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohortsis determined using receiver operating characteristic (ROC) analysis.

TABLE 16 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 24 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 6.84 40.0 11.7 34.1 10.1 47.0Average 22.5 72.8 32.2 78.4 35.3 81.4 Stdev 39.0 84.4 45.0 92.4 61.684.3 p (t-test) 5.4E−5 1.3E−4 1.7E−4 Min 0.0130 0.0690 0.0130 0.06900.0130 0.129 Max 223 438 223 438 438 338 n (Patient) 55 94 78 71 88 61sCr only Median 6.92 35.3 11.7 35.3 9.80 47.5 Average 24.3 72.3 30.578.4 34.1 83.5 Stdev 39.6 85.7 42.5 92.6 60.1 85.6 p (t-test) 1.1E−47.5E−5 6.2E−5 Min 0.0130 0.0690 0.0130 0.0690 0.0130 0.129 Max 223 438223 438 438 338 n (Patient) 57 91 76 72 89 59 UO only Median 14.1 70.314.5 95.8 15.0 95.8 Average 45.4 85.2 45.5 95.4 47.6 92.5 Stdev 69.884.9 68.5 90.4 71.3 85.4 p (t-test) 0.0063 0.0016 0.0081 Min 0.01300.819 0.0130 0.819 0.0130 0.819 Max 438 338 438 338 438 338 n (Patient)114 34 121 27 125 23 Persistence Period Duration (hr) 24 48 72 sCr orsCr or sCr or UO sCr only UO only UO sCr only UO only UO sCr only UOonly AUC 0.73 0.70 0.68 0.66 0.66 0.69 0.70 0.70 0.69 SE 0.041 0.0420.055 0.045 0.045 0.060 0.045 0.045 0.065 p Value 2.7E−8 1.7E−6 0.00122.4E−4 2.9E−4 0.0014 7.6E−6 5.6E−6 0.0045 nCohort Non-persistent 55 57114 78 76 121 88 89 125 nCohort Persistent 94 91 34 71 72 27 61 59 23Cutoff Quartile 2 3.43 3.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56Sensitivity 85% 84% 94% 85% 83% 93% 89% 88% 91% Specificity 42% 39% 31%33% 33% 29% 34% 34% 28% Cutoff Quartile 3 17.6 16.9 17.8 17.6 16.9 17.817.6 16.9 17.8 Sensitivity 64% 63% 71% 63% 62% 74% 69% 69% 74%Specificity 73% 70% 56% 62% 62% 55% 62% 63% 54% Cutoff Quartile 4 89.088.4 89.4 89.0 88.4 89.4 89.0 88.4 89.4 Sensitivity 36% 35% 50% 42% 42%56% 46% 46% 57% Specificity 93% 91% 82% 90% 91% 82% 89% 89% 81% ORQuartile 2 4.11 3.18 7.09 2.73 2.45 5.09 3.99 3.78 4.08 p Value 3.9E−40.0031 0.0096 0.014 0.025 0.033 0.0026 0.0039 0.066 Lower limit of 95%CI 1.88 1.48 1.61 1.23 1.12 1.14 1.62 1.53 0.909 Upper limit of 95% CI8.97 6.87 31.2 6.05 5.36 22.6 9.84 9.32 18.3 OR Quartile 3 4.71 3.943.07 2.77 2.70 3.54 3.68 3.87 3.38 p Value 3.0E−5 1.5E−4 0.0077 0.00270.0034 0.0078 2.2E−4 1.6E−4 0.016 Lower limit of 95% CI 2.27 1.94 1.351.43 1.39 1.40 1.84 1.92 1.25 Upper limit of 95% CI 9.74 8.01 7.01 5.385.25 9.01 7.37 7.80 9.14 OR Quartile 4 7.22 5.64 4.70 6.40 7.04 5.626.62 6.67 5.47 p Value 4.3E−4 8.2E−4 2.5E−4 2.9E−5 2.5E−5 1.4E−4 7.9E−68.2E−6 3.8E−4 Lower limit of 95% CI 2.40 2.05 2.05 2.68 2.84 2.31 2.892.90 2.14 Upper limit of 95% CI 21.7 15.5 10.8 15.3 17.5 13.7 15.2 15.314.0

TABLE 17 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 48 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 6.65 43.6 9.80 46.5 9.29 55.6Average 20.7 73.2 26.2 81.1 25.3 89.6 Stdev 37.2 84.1 38.3 90.5 37.692.5 p (t-test) 2.6E−5 4.1E−6 5.1E−8 Min 0.0130 0.0690 0.0130 0.06900.0130 0.129 Max 223 438 223 438 223 438 n (Patient) 54 95 73 76 82 67sCr only Median 6.88 35.9 10.4 36.4 9.29 49.1 Average 22.7 72.8 27.579.4 26.4 87.9 Stdev 37.9 85.3 39.0 91.5 38.4 93.8 p (t-test) 5.6E−51.6E−5 2.6E−7 Min 0.0130 0.0690 0.0130 0.0690 0.0130 0.129 Max 223 438223 438 223 438 n (Patient) 56 92 73 75 82 66 UO only Median 13.5 52.013.6 92.1 13.6 92.1 Average 40.9 89.0 41.3 97.6 43.7 95.7 Stdev 59.797.1 58.7 103 62.7 101 p (t-test) 3.6E−4 7.6E−5 5.0E−4 Min 0.0130 0.8190.0130 0.819 0.0130 0.819 Max 237 438 237 438 298 438 n (Patient) 106 42113 35 117 31 Persistence Period Duration (hr) 24 48 72 sCr or sCr orsCr or UO sCr only UO only UO sCr only UO only UO sCr only UO only AUC0.74 0.71 0.70 0.71 0.68 0.70 0.75 0.73 0.69 SE 0.040 0.042 0.051 0.0420.044 0.054 0.041 0.042 0.057 p Value 2.7E−9 3.1E−7 1.1E−4 8.5E−7 2.2E−51.6E−4 4.9E−10 3.7E−8 6.7E−4 nCohort Non-persistent 54 56 106 73 73 11382 82 117 nCohort Persistent 95 92 42 76 75 35 67 66 31 Cutoff Quartile2 3.43 3.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56 Sensitivity 85% 84% 93%86% 84% 91% 90% 88% 90% Specificity 43% 39% 32% 36% 34% 30% 37% 35% 29%Cutoff Quartile 3 17.6 16.9 17.8 17.6 16.9 17.8 17.6 16.9 17.8Sensitivity 64% 63% 71% 66% 64% 74% 72% 70% 74% Specificity 74% 71% 58%66% 64% 58% 67% 66% 56% Cutoff Quartile 4 89.0 88.4 89.4 89.0 88.4 89.489.0 88.4 89.4 Sensitivity 37% 36% 48% 45% 43% 51% 49% 47% 52%Specificity 94% 93% 84% 95% 93% 83% 94% 93% 82% OR Quartile 2 4.29 3.326.14 3.27 2.73 4.59 4.95 3.97 3.82 p Value 2.6E−4 0.0023 0.0042 0.00370.012 0.017 5.2E−4 0.0018 0.036 Lower limit of 95% CI 1.96 1.54 1.771.47 1.25 1.32 2.01 1.67 1.09 Upper limit of 95% CI 9.39 7.18 21.3 7.275.99 16.0 12.2 9.44 13.4 OR Quartile 3 5.13 4.26 3.52 3.69 3.21 3.915.15 4.44 3.72 p Value 1.5E−5 7.5E−5 0.0014 1.6E−4 6.6E−4 0.0016 5.0E−62.7E−5 0.0036 Lower limit of 95% CI 2.45 2.08 1.63 1.88 1.64 1.68 2.552.21 1.54 Upper limit of 95% CI 10.7 8.74 7.63 7.27 6.29 9.11 10.4 8.899.00 OR Quartile 4 9.92 7.27 4.76 14.0 10.1 5.24 14.9 11.2 4.88 p Value2.8E−4 4.2E−4 1.3E−4 2.9E−6 8.2E−6 8.5E−5 2.2E−7 8.3E−7 2.5E−4 Lowerlimit of 95% CI 2.88 2.41 2.14 4.63 3.66 2.29 5.37 4.29 2.09 Upper limitof 95% CI 34.2 21.9 10.6 42.2 28.0 12.0 41.6 29.3 11.4

TABLE 18 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 72 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 6.65 43.6 9.56 46.1 9.29 47.5Average 20.7 73.2 26.3 80.3 26.2 85.0 Stdev 37.2 84.1 38.5 90.3 38.491.9 p (t-test) 2.6E−5 6.3E−6 7.3E−7 Min 0.0130 0.0690 0.0130 0.06900.0130 0.129 Max 223 438 223 438 223 438 n (Patient) 54 95 72 77 78 71sCr only Median 6.88 35.9 10.1 35.3 9.56 46.5 Average 22.7 72.8 27.778.6 27.4 83.2 Stdev 37.9 85.3 39.3 91.2 39.0 93.0 p (t-test) 5.6E−52.4E−5 3.2E−6 Min 0.0130 0.0690 0.0130 0.0690 0.0130 0.129 Max 223 438223 438 223 438 n (Patient) 56 92 72 76 78 70 UO only Median 14.1 41.314.1 46.8 14.7 41.3 Average 43.0 78.7 42.7 84.6 45.2 81.8 Stdev 60.894.8 59.7 99.0 63.8 97.0 p (t-test) 0.0063 0.0019 0.0092 Min 0.01300.819 0.0130 0.819 0.0130 0.819 Max 237 438 237 438 298 438 n (Patient)100 48 106 42 110 38 Persistence Period Duration (hr) 24 48 72 sCr orsCr or sCr or UO sCr only UO only UO sCr only UO only UO sCr only UOonly AUC 0.74 0.71 0.64 0.71 0.68 0.65 0.73 0.71 0.64 SE 0.040 0.0420.050 0.042 0.043 0.052 0.042 0.043 0.054 p Value 2.7E−9 3.1E−7 0.00419.2E−7 2.3E−5 0.0040 3.2E−8 1.5E−6 0.012 nCohort Non-persistent 54 56100 72 72 106 78 78 110 nCohort Persistent 95 92 48 77 76 42 71 70 38Cutoff Quartile 2 3.43 3.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56Sensitivity 85% 84% 88% 86% 84% 86% 89% 87% 84% Specificity 43% 39% 31%36% 35% 29% 37% 36% 28% Cutoff Quartile 3 17.6 16.9 17.8 17.6 16.9 17.817.6 16.9 17.8 Sensitivity 64% 63% 65% 65% 63% 67% 68% 66% 66%Specificity 74% 71% 57% 65% 64% 57% 65% 64% 55% Cutoff Quartile 4 89.088.4 89.4 89.0 88.4 89.4 89.0 88.4 89.4 Sensitivity 37% 36% 42% 44% 42%45% 46% 44% 45% Specificity 94% 93% 83% 94% 93% 83% 94% 92% 82% ORQuartile 2 4.29 3.32 3.14 3.39 2.84 2.48 4.66 3.80 2.09 p Value 2.6E−40.0023 0.019 0.0027 0.0092 0.064 5.0E−4 0.0018 0.13 Lower limit of 95%CI 1.96 1.54 1.21 1.53 1.29 0.949 1.96 1.64 0.797 Upper limit of 95% CI9.39 7.18 8.17 7.54 6.22 6.48 11.1 8.78 5.50 OR Quartile 3 5.13 4.262.42 3.48 3.03 2.61 3.94 3.42 2.39 p Value 1.5E−5 7.5E−5 0.015 2.9E−40.0012 0.012 8.0E−5 3.6E−4 0.026 Lower limit of 95% CI 2.45 2.08 1.191.77 1.55 1.24 1.99 1.74 1.11 Upper limit of 95% CI 10.7 8.74 4.93 6.835.93 5.51 7.79 6.73 5.16 OR Quartile 4 9.92 7.27 3.49 13.4 9.75 4.0412.7 9.54 3.64 p Value 2.8E−4 4.2E−4 0.0016 4.0E−6 1.1E−5 5.5E−4 1.0E−63.9E−6 0.0016 Lower limit of 95% CI 2.88 2.41 1.61 4.46 3.53 1.83 4.583.66 1.63 Upper limit of 95% CI 34.2 21.9 7.57 40.6 26.9 8.91 35.1 24.88.13

TABLE 19 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 96 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 6.65 43.6 9.56 46.1 9.29 47.5Average 20.7 73.2 26.3 80.3 26.2 85.0 Stdev 37.2 84.1 38.5 90.3 38.491.9 p (t-test) 2.6E−5 6.3E−6 7.3E−7 Min 0.0130 0.0690 0.0130 0.06900.0130 0.129 Max 223 438 223 438 223 438 n (Patient) 54 95 72 77 78 71sCr only Median 6.88 35.9 10.1 35.3 9.56 46.5 Average 22.7 72.8 27.778.6 27.4 83.2 Stdev 37.9 85.3 39.3 91.2 39.0 93.0 p (t-test) 5.6E−52.4E−5 3.2E−6 Min 0.0130 0.0690 0.0130 0.0690 0.0130 0.129 Max 223 438223 438 223 438 n (Patient) 56 92 72 76 78 70 UO only Median 13.6 41.313.6 46.1 14.5 36.4 Average 43.2 76.9 43.2 80.6 45.8 77.6 Stdev 61.493.4 60.5 96.9 64.6 94.6 p (t-test) 0.0095 0.0049 0.021 Min 0.0130 0.8190.0130 0.819 0.0130 0.819 Max 237 438 237 438 298 438 n (Patient) 98 50103 45 107 41 Persistence Period Duration (hr) 24 48 72 sCr or sCr orsCr or UO sCr only UO only UO sCr only UO only UO sCr only UO only AUC0.74 0.71 0.64 0.71 0.68 0.64 0.73 0.71 0.63 SE 0.040 0.042 0.049 0.0420.043 0.051 0.042 0.043 0.053 p Value 2.7E−9 3.1E−7 0.0034 9.2E−7 2.3E−50.0048 3.2E−8 1.5E−6 0.015 nCohort Non-persistent 54 56 98 72 72 103 7878 107 nCohort Persistent 95 92 50 77 76 45 71 70 41 Cutoff Quartile 23.43 3.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56 Sensitivity 85% 84% 88% 86%84% 87% 89% 87% 85% Specificity 43% 39% 32% 36% 35% 30% 37% 36% 29%Cutoff Quartile 3 17.6 16.9 17.8 17.6 16.9 17.8 17.6 16.9 17.8Sensitivity 64% 63% 64% 65% 63% 64% 68% 66% 63% Specificity 74% 71% 57%65% 64% 56% 65% 64% 55% Cutoff Quartile 4 89.0 88.4 89.4 89.0 88.4 89.489.0 88.4 89.4 Sensitivity 37% 36% 40% 44% 42% 42% 46% 44% 41%Specificity 94% 93% 83% 94% 93% 83% 94% 92% 81% OR Quartile 2 4.29 3.323.39 3.39 2.84 2.80 4.66 3.80 2.38 p Value 2.6E−4 0.0023 0.012 0.00270.0092 0.035 5.0E−4 0.0018 0.077 Lower limit of 95% CI 1.96 1.54 1.311.53 1.29 1.07 1.96 1.64 0.910 Upper limit of 95% CI 9.39 7.18 8.80 7.546.22 7.29 11.1 8.78 6.22 OR Quartile 3 5.13 4.26 2.37 3.48 3.03 2.343.94 3.42 2.13 p Value 1.5E−5 7.5E−5 0.016 2.9E−4 0.0012 0.022 8.0E−53.6E−4 0.045 Lower limit of 95% CI 2.45 2.08 1.17 1.77 1.55 1.13 1.991.74 1.02 Upper limit of 95% CI 10.7 8.74 4.79 6.83 5.93 4.82 7.79 6.734.47 OR Quartile 4 9.92 7.27 3.18 13.4 9.75 3.45 12.7 9.54 3.08 p Value2.8E−4 4.2E−4 0.0033 4.0E−6 1.1E−5 0.0019 1.0E−6 3.9E−6 0.0052 Lowerlimit of 95% CI 2.88 2.41 1.47 4.46 3.53 1.58 4.58 3.66 1.40 Upper limitof 95% CI 34.2 21.9 6.86 40.6 26.9 7.53 35.1 24.8 6.78

TABLE 20 Comparison of marker levels and the area under the ROC curve(AUC) in urine samples for the “persistent” and “non-persistent” cohortswhere persistence starts within 168 hours after sample collection andrenal status is assessed by serum creatinine (sCr) only, urine output(UO) only, or serum creatinine or urine output RIFLE criteria.Persistence Period Duration (hr) 24 48 72 Non-persistent PersistentNon-persistent Persistent Non-persistent Persistent Cohort Cohort CohortCohort Cohort Cohort sCr or UO Median 6.65 43.6 9.80 41.3 9.29 47.0Average 19.8 72.7 26.7 77.9 26.4 81.6 Stdev 36.5 83.6 39.1 89.4 39.190.6 p (t-test) 2.6E−5 2.0E−5 3.6E−6 Min 0.0130 0.0690 0.0130 0.06900.0130 0.129 Max 223 438 223 438 223 438 n (Patient) 52 97 69 80 74 75sCr only Median 6.84 36.4 9.56 41.3 9.29 47.3 Average 21.4 73.0 26.578.4 26.3 82.9 Stdev 37.1 84.9 39.0 90.1 38.8 91.8 p (t-test) 3.6E−51.6E−5 2.3E−6 Min 0.0130 0.0690 0.0130 0.0690 0.0130 0.129 Max 223 438223 438 223 438 n (Patient) 55 93 70 78 76 72 UO only Median 13.5 46.513.6 46.1 13.6 41.3 Average 42.1 77.7 42.6 78.8 42.8 80.7 Stdev 61.192.1 60.6 94.2 60.1 96.5 p (t-test) 0.0055 0.0053 0.0041 Min 0.01300.819 0.0130 0.819 0.0130 0.819 Max 237 438 237 438 237 438 n (Patient)96 52 99 49 102 46 Persistence Period Duration (hr) 24 48 72 sCr or sCror sCr or UO sCr only UO only UO sCr only UO only UO sCr only UO onlyAUC 0.74 0.72 0.66 0.69 0.70 0.65 0.72 0.72 0.65 SE 0.040 0.041 0.0480.043 0.043 0.049 0.042 0.042 0.050 p Value 4.1E−9 6.6E−8 0.0011 5.5E−64.9E−6 0.0025 1.9E−7 2.6E−7 0.0032 nCohort Non-persistent 52 55 96 69 7099 74 76 102 nCohort Persistent 97 93 52 80 78 49 75 72 46 CutoffQuartile 2 3.43 3.42 3.56 3.43 3.42 3.56 3.43 3.42 3.56 Sensitivity 85%84% 88% 85% 85% 88% 88% 88% 87% Specificity 42% 40% 32% 36% 36% 31% 38%37% 30% Cutoff Quartile 3 17.6 16.9 17.8 17.6 16.9 17.8 17.6 16.9 17.8Sensitivity 64% 63% 65% 64% 64% 65% 67% 67% 65% Specificity 75% 73% 58%65% 66% 58% 66% 66% 57% Cutoff Quartile 4 89.0 88.4 89.4 89.0 88.4 89.489.0 88.4 89.4 Sensitivity 37% 37% 40% 42% 42% 41% 44% 44% 41%Specificity 96% 95% 83% 94% 94% 83% 93% 93% 82% OR Quartile 2 4.01 3.473.66 3.22 3.06 3.27 4.46 4.08 2.91 p Value 4.7E−4 0.0016 0.0076 0.00350.0053 0.015 4.8E−4 0.0010 0.029 Lower limit of 95% CI 1.84 1.60 1.411.47 1.39 1.26 1.93 1.76 1.12 Upper limit of 95% CI 8.73 7.50 9.48 7.076.70 8.48 10.3 9.46 7.57 OR Quartile 3 5.31 4.63 2.64 3.30 3.42 2.553.92 3.85 2.47 p Value 1.3E−5 3.7E−5 0.0065 5.1E−4 3.6E−4 0.0097 8.3E−51.1E−4 0.014 Lower limit of 95% CI 2.50 2.23 1.31 1.68 1.74 1.26 1.991.95 1.20 Upper limit of 95% CI 11.3 9.58 5.33 6.46 6.73 5.20 7.74 7.605.09 OR Quartile 4 14.8 9.99 3.39 12.0 12.1 3.33 10.8 11.4 3.28 p Value3.4E−4 2.7E−4 0.0019 9.9E−6 9.7E−6 0.0023 4.2E−6 3.0E−6 0.0027 Lowerlimit of 95% CI 3.38 2.90 1.57 3.99 4.01 1.54 3.93 4.10 1.51 Upper limitof 95% CI 64.3 34.4 7.33 36.2 36.5 7.21 29.9 31.5 7.14

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method for evaluating renal status in a subjectthat meets the definition of RIFLE I or F for persistence of acute renalinjury, comprising: performing an assay method configured to detectChitinase-3-like protein 1 on a body fluid sample obtained from thesubject to provide an assay result by introducing the body fluid sampleobtained from the subject into an assay instrument which (i) contactsall or a portion of the body fluid sample with a binding reagent whichspecifically binds for detection Chitinase-3-like protein 1, and (ii)generates an assay result indicative of binding of Chitinase-3-likeprotein 1 to its binding reagent; correlating the assay result to therenal status of the subject by using the assay result to assign thepatient to a predetermined subpopulation of individuals having a knownlikelihood of recovery from acute renal injury within 72 hours of thetime at which the body fluid is obtained, the assignment made bycomparing the assay result or a value derived therefrom to a thresholdvalue obtained from a population study, wherein the threshold separatesthe population into a first subpopulation above the threshold and asecond subpopulation below the threshold, the second subpopulation beingat an increased likelihood persistent acute renal injury relative to thefirst subpopulation; and treating the patient based on the predeterminedsubpopulation of individuals to which the patient is assigned, whereinwhen the assay result or value derived therefrom assigns the subject tothe second subpopulation, the subject is treated for persistent acuterenal injury by initiation or continuation of renal replacement therapy.2-4. (canceled)
 5. A method according to claim 1, wherein said assayresults comprise a measured concentration of Chitinase-3-like protein 1.6. A method according to claim 1, wherein said correlating stepcomprises combining a plurality of assay results, one of which is theChitinase-3-like protein 1 assay result, using a function that convertsthe plurality of assay results into a single composite result. 7-108.(canceled)
 109. A method according to claim 1, wherein the subject meetsthe definition of RIFLE F.